Bio

Bio

Sharon Long received her undergraduate degree from Caltech, and carried out her PhD studies at Yale, working with Ian Sussex on plant development. She was a postdoc with Fred Ausubel where she began study of rhizobia-legume symbioses. She joined the Stanford faculty in 1982.

Abstract

Sinorhizobium meliloti is a soil-dwelling ?-proteobacterium that possesses a large, tripartite genome and engages in a nitrogen fixing symbiosis with its plant hosts. Although much is known about this important model organism, global characterization of genetic regulatory circuits has been hampered by a lack of information about transcription and promoters.Using an RNAseq approach and RNA populations representing 16 different growth and stress conditions, we comprehensively mapped S. meliloti transcription start sites (TSS). Our work identified 17,001 TSS that we grouped into six categories based on the genomic context of their transcripts: mRNA (4,430 TSS assigned to 2,657 protein-coding genes), leaderless mRNAs (171), putative mRNAs (425), internal sense transcripts (7,650), antisense RNA (3,720), and trans-encoded sRNAs (605). We used this TSS information to identify transcription factor binding sites and putative promoter sequences recognized by seven of the 15 known S. meliloti ? factors ?70, ?54, ?H1, ?H2, ?E1, ?E2, and ?E9). Altogether, we predicted 2,770 new promoter sequences, including 1,302 located upstream of protein coding genes and 722 located upstream of antisense RNA or trans-encoded sRNA genes. To validate promoter predictions for targets of the general stress response ? factor, RpoE2 (?E2), we identified rpoE2-dependent genes using microarrays and confirmed TSS for a subset of these by 5' RACE mapping.By identifying TSS and promoters on a global scale, our work provides a firm foundation for the continued study of S. meliloti gene expression with relation to gene organization, ? factors and other transcription factors, and regulatory RNAs.

Abstract

The Medicago truncatula DMI2 gene encodes a leucine-rich repeat receptor-like kinase that is essential for symbiosis with nitrogen-fixing rhizobia. While phenotypic analyses have provided a description for the host's responses mediated by DMI2, a lack of tools for in vivo biochemical analysis has hampered efforts to elucidate the mechanisms by which DMI2 mediates symbiotic signal transduction. Here, we report stably transformed M. truncatula lines that express a genomic DMI2 construct that is fused to a dual-affinity tag containing three copies of the hemagglutinin epitope and a single StrepII tag (gDMI2:HAST). gDMI2: HAST complements the dmi2-1 mutation, and transgenic plants expressing this construct behave similarly to wild-type plants. We show that the expression patterns of gDMI2:HAST recapitulate those of endogenous DMI2 and that we can detect and purify DMI2:HAST from microsomal root and nodule extracts. Using this line, we show that DMI2 resides in a high-molecular weight complex, which is consistent with our observation that DMI2:GFP localizes to plasma membrane-associated puncta and cytoplasmic vesicles. We further demonstrate that Nod factor (NF) perception increases the abundance of DMI2 vesicles. These tools should be a valuable resource for the Medicago community to dissect the biochemical function of DMI2.

Abstract

Sinorhizobium meliloti can live as a soil saprophyte and can engage in a nitrogen-fixing symbiosis with plant roots. To succeed in such diverse environments, the bacteria must continually adjust gene expression. Transcriptional plasticity in eubacteria is often mediated by alternative sigma (?) factors interacting with core RNA polymerase. The S. meliloti genome encodes 14 of these alternative ? factors, including two putative RpoH ("heat shock") ? factors. We used custom Affymetrix symbiosis chips to characterize the global transcriptional response of S. meliloti rpoH1, rpoH2, and rpoH1 rpoH2 mutants during heat shock and stationary-phase growth. Under these conditions, expression of over 300 genes is dependent on rpoH1 and rpoH2. We mapped transcript start sites of 69 rpoH-dependent genes using 5' RACE (5' rapid amplification of cDNA ends), which allowed us to determine putative RpoH1-dependent, RpoH2-dependent, and dual-promoter (RpoH1- and RpoH2-dependent) consensus sequences that were each used to search the genome for other potential direct targets of RpoH. The inferred S. meliloti RpoH promoter consensus sequences share features of Escherichia coli RpoH promoters but lack extended -10 motifs.

Abstract

The genetic rules that dictate legume-rhizobium compatibility have been investigated for decades, but the causes of incompatibility occurring at late stages of the nodulation process are not well understood. An evaluation of naturally diverse legume (genus Medicago) and rhizobium (genus Sinorhizobium) isolates has revealed numerous instances in which Sinorhizobium strains induce and occupy nodules that are only minimally beneficial to certain Medicago hosts. Using these ineffective strain-host pairs, we identified gain-of-compatibility (GOC) rhizobial variants. We show that GOC variants arise by loss of specific large accessory plasmids, which we call HR plasmids due to their effect on symbiotic host range. Transfer of HR plasmids to a symbiotically effective rhizobium strain can convert it to incompatibility, indicating that HR plasmids can act autonomously in diverse strain backgrounds. We provide evidence that HR plasmids may encode machinery for their horizontal transfer. On hosts in which HR plasmids impair N fixation, the plasmids also enhance competitiveness for nodule occupancy, showing that naturally occurring, transferrable accessory genes can convert beneficial rhizobia to a more exploitative lifestyle. This observation raises important questions about agricultural management, the ecological stability of mutualisms, and the genetic factors that distinguish beneficial symbionts from parasites.

Abstract

Although diminutive in size, bacteria possess highly diverse and spatially confined cellular structures. Two related alphaproteobacteria, Sinorhizobium meliloti and Caulobacter crescentus, serve as models for investigating the genetic basis of morphological variations. S. meliloti, a symbiont of leguminous plants, synthesizes multiple flagella and no prosthecae, whereas C. crescentus, a freshwater bacterium, has a single polar flagellum and stalk. The podJ gene, originally identified in C. crescentus for its role in polar organelle development, is split into two adjacent open reading frames, podJ1 and podJ2, in S. meliloti. Deletion of podJ1 interferes with flagellar motility, exopolysaccharide production, cell envelope integrity, cell division and normal morphology, but not symbiosis. As in C. crescentus, the S. meliloti PodJ1 protein appears to act as a polarity beacon and localizes to the newer cell pole. Microarray analysis indicates that podJ1 affects the expression of at least 129 genes, the majority of which correspond to observed mutant phenotypes. Together, phenotypic characterization, microarray analysis and suppressor identification suggest that PodJ1 controls a core set of conserved elements, including flagellar and pili genes, the signalling proteins PleC and DivK, and the transcriptional activator TacA, while alternative downstream targets have evolved to suit the distinct lifestyles of individual species.

Abstract

Rhizobium and allied bacteria form symbiotic nitrogen-fixing nodules on legume roots. Plant hormones play key roles in nodule formation. We treated Medicago truncatula roots with auxin transport inhibitors (ATI) N-(1-naphthyl)phthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA) to induce the formation of pseudonodules. M. truncatula mutants defective for rhizobial Nod factor signal transduction still formed pseudonodules in response to ATI. However, a M. truncatula ethylene-insensitive supernodulator, sickle 1-1, did not form pseudonodules in response to TIBA, suggesting that the ethylene response pathway is involved in ATI-induced pseudonodule formation. We compared the transcriptional responses of M. truncatula roots treated with ATI to roots inoculated with Sinorhizobium meliloti. Some genes showed consistently parallel expression in ATI-induced and Rhizobium-induced nodules. For other genes, the transcriptional response of M. truncatula roots 1 and 7 days after ATI treatment was in the opposite direction to roots treated with S. meliloti; then, by 21 days, the transcriptional patterns for the two conditions became similar. We silenced 17 genes that were upregulated in both ATI and S. meliloti treatments to determine their effect on nodule formation. Some gene-silenced roots showed a decrease in nodulation efficiency, suggesting a role in nodule formation but not in later nodule functions.

Abstract

The formation of nitrogen-fixing nodules in legumes is tightly controlled by a long-distance signaling system in which nodulating roots signal to shoot tissues to suppress further nodulation. A screen for supernodulating Medicago truncatula mutants defective in this regulatory behavior yielded loss-of-function alleles of a gene designated ROOT DETERMINED NODULATION1 (RDN1). Grafting experiments demonstrated that RDN1 regulatory function occurs in the roots, not the shoots, and is essential for normal nodule number regulation. The RDN1 gene, Medtr5g089520, was identified by genetic mapping, transcript profiling, and phenotypic rescue by expression of the wild-type gene in rdn1 mutants. A mutation in a putative RDN1 ortholog was also identified in the supernodulating nod3 mutant of pea (Pisum sativum). RDN1 is predicted to encode a 357-amino acid protein of unknown function. The RDN1 promoter drives expression in the vascular cylinder, suggesting RDN1 may be involved in initiating, responding to, or transporting vascular signals. RDN1 is a member of a small, uncharacterized, highly conserved gene family unique to green plants, including algae, that we have named the RDN family.

Abstract

To form nitrogen-fixing symbioses, legume plants recognize a bacterial signal, Nod Factor (NF). The legume Medicago truncatula has two predicted NF receptors that direct separate downstream responses to its symbiont Sinorhizobium meliloti. NOD FACTOR PERCEPTION encodes a putative low-stringency receptor that is responsible for calcium spiking and transcriptional responses. LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) encodes a putative high-stringency receptor that mediates bacterial infection. We localized green fluorescent protein (GFP)-tagged LYK3 in M. truncatula and found that it has a punctate distribution at the cell periphery consistent with a plasma membrane or membrane-tethered vesicle localization. In buffer-treated control roots, LYK3:GFP puncta are dynamic. After inoculation with compatible S. meliloti, LYK3:GFP puncta are relatively stable. We show that increased LYK3:GFP stability depends on bacterial NF and NF structure but that NF is not sufficient for the change in LYK3:GFP dynamics. In uninoculated root hairs, LYK3:GFP has little codistribution with mCherry-tagged FLOTILLIN4 (FLOT4), another punctate plasma membrane-associated protein required for infection. In inoculated root hairs, we observed an increase in FLOT4:mCherry and LYK3:GFP colocalization; both proteins localize to positionally stable puncta. We also demonstrate that the localization of tagged FLOT4 is altered in plants carrying a mutation that inactivates the kinase domain of LYK3. Our work indicates that LYK3 protein localization and dynamics are altered in response to symbiotic bacteria.

Abstract

The ability to remove a genetic function from an organism with good temporal resolution is crucial for characterizing essential genes or genes that act in complex developmental programs. The rhizobium-legume symbiosis involves an elaborate two-organism interaction requiring multiple levels of signal exchange. As an important step toward probing rhizobium genetic functions with temporal resolution, we present the development of a conditional gene deletion system in Sinorhizobium meliloti that employs Cre/loxP site-specific recombination. This system enables chemically inducible and irreversible gene deletion or gene upregulation. Recombinase-mediated excision events can be positively or negatively selected or monitored by a colorimetric assay. The system may be adaptable to various bacterial species, in which recombinase activity may be placed under the control of diverse user-defined promoters. This system also shows promise for uses in promoter trapping and biosensing applications.

Abstract

One hundred years ago, Flexner emphasized the importance of science in medicine and medical education. Over the subsequent years, science education in the premedical and medical curricula has changed little, in spite of the vast changes in the biomedical sciences. The National Research Council, in their report Bio 2010, noted that the premedical curriculum caused many students to lose interest in medicine and in the biological sciences in general. Many medical students and physicians have come to view the premedical curriculum as of limited relevance to medicine and designed more as a screening mechanism for medical school admission. To address this, the Association of American Medical Colleges and the Howard Hughes Medical Institute formed a committee to evaluate the premedical and medical school science curricula. The committee made a number of recommendations that are summarized in this essay. Most important were that competencies replace course requirements and that the physical sciences and mathematics be better integrated with the biological sciences and medicine. The goal is that all physicians possess a strong scientific knowledge base and come to appreciate the importance of this to the practice of medicine. While science education needs to evolve, Flexner's vision is as relevant today as it was 100 years ago.

Abstract

Nitrogen-fixing symbioses of plants are often associated with bacterially infected nodules where nitrogen fixation occurs. The plant host facilitates bacterial infection with the formation of infection threads, unique structures associated with these symbioses, which are invaginations of the host cell with the capability of traversing cellular junctions. Here, we show that the infection thread shares mechanistic similarities to polar-growing cells, because the required for infection thread (RIT) locus of Medicago truncatula has roles in root-hair, trichome, and infection-thread growth. We show that RIT encodes the M. truncatula ortholog of NAP1, a component of the SCAR/WAVE (suppressor of cAMP receptor/WASP-family verprolin homologous protein) complex that regulates actin polymerization, through the activation of ARP2/3. NAP1 of Arabidopsis thaliana functions equivalently to the M. truncatula gene, indicating that the mode of action of NAP1 is functionally conserved across species and that legumes have not evolved a unique functionality for NAP1 during rhizobial colonization. This work highlights the surprising commonality between polar-growing cells and a polar-growing cellular intrusion and reveals important insights into the formation and maintenance of infection-thread development.

Abstract

A novel autoregulation of nodulation locus in Medicago truncatula, lss, silences the SUNN gene thorough a cis-acting mechanism. Microarray analysis was performed on the Affymetrix Gene Chip® Medicago Genome Array with cDNA isolated from seven-day-old seedlings of wild type, sunn-1 and lss plants. The results suggest that in lss plants expression of only a few dozen genes differs significantly from wild type while in sunn-1 plants expression of several hundred genes represented by over 800 probe sets is altered. These results suggest that the kinase domain modification caused by the sunn-1 mutation alters the receptor's influence on gene expression and that these differences are present even in the absence of nodulation.

Abstract

The number of nodules that form in a legume when interacting with compatible rhizobia is regulated by the plant. We report the identification of a mutant in nodule regulation in Medicago truncatula, like sunn supernodulator (lss), which displays shoot-controlled supernodulation and short roots, similar to sunn mutants. In contrast with the sunn-1 mutant, nodulation in the lss mutant is more extensive and is less sensitive to nitrate and ethylene, resembling the sunn-4 presumed null allele phenotype. Although the lss locus maps to the SUNN region of linkage group 4 and sunn and lss do not complement each other, there is no mutation in the genomic copy of the SUNN gene or in the 15-kb surrounding region in the lss mutant. However, expression of the SUNN gene in the shoots of lss plants is greatly reduced compared with wild-type plants. Analysis of cDNA from plants heterozygous for lss indicates that lss is a cis-acting factor affecting the expression of SUNN, and documented reversion events show it to be unstable, suggesting a possible reversible DNA rearrangement or an epigenetic change in the lss mutant. Assessment of the SUNN promoter revealed low levels of cytosine methylation in the 700-bp region proximal to the predicted transcription start site in both wild-type and lss plants, indicating that promoter hypermethylation is not responsible for the suppression of SUNN expression in lss. Thus, lss represents either a distal novel locus within the mapped region affecting SUNN expression or an uncharacterized epigenetic modification at the SUNN locus.

Abstract

The RNA-binding protein Hfq is a global regulator which controls diverse cellular processes in bacteria. To begin understanding the role of Hfq in the Sinorhizobium meliloti-Medicago truncatula nitrogen-fixing symbiosis, we defined free-living and symbiotic phenotypes of an hfq mutant. Over 500 transcripts were differentially accumulated in the hfq mutant of S. meliloti Rm1021 when grown in a shaking culture. Consistent with transcriptome-wide changes, the hfq mutant displayed dramatic alterations in metabolism of nitrogen-containing compounds, even though its carbon source utilization profiles were nearly identical to the wild type. The hfq mutant had reduced motility and was impaired for growth at alkaline pH. A deletion of hfq resulted in a reduced symbiotic efficiency, although the mutant was still able to initiate nodule development and differentiate into bacteroids.

Abstract

The nitrogen-fixing symbiosis between Sinorhizobium meliloti and its leguminous host plant Medicago truncatula occurs in a specialized root organ called the nodule. Bacteria that are released into plant cells are surrounded by a unique plant membrane compartment termed a symbiosome. We found that in the symbiosis-defective dnf1 mutant of M. truncatula, bacteroid and symbiosome development are blocked. We identified the DNF1 gene as encoding a subunit of a signal peptidase complex that is highly expressed in nodules. By analyzing data from whole-genome expression analysis, we propose that correct symbiosome development in M. truncatula requires the orderly secretion of protein constituents through coordinated up-regulation of a nodule-specific pathway exemplified by DNF1.

Plant flotillins are required for infection by nitrogen-fixing bacteriaPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAHaney, C. H., Long, S. R.2010; 107 (1): 478-483

Abstract

To establish compatible rhizobial-legume symbioses, plant roots support bacterial infection via host-derived infection threads (ITs). Here, we report the requirement of plant flotillin-like genes (FLOTs) in Sinorhizobium meliloti infection of its host legume Medicago truncatula. Flotillins in other organisms have roles in viral pathogenesis, endocytosis, and membrane shaping. We identified seven FLOT genes in the M. truncatula genome and show that two, FLOT2 and FLOT4, are strongly up-regulated during early symbiotic events. This up-regulation depends on bacterial Nod Factor and the plant's ability to perceive Nod Factor. Microscopy data suggest that M. truncatula FLOT2 and FLOT4 localize to membrane microdomains. Upon rhizobial inoculation, FLOT4 uniquely becomes localized to the tips of elongating root hairs. Silencing FLOT2 and FLOT4 gene expression reveals a nonredundant requirement for both genes in IT initiation and nodule formation. FLOT4 is uniquely required for IT elongation, and FLOT4 localizes to IT membranes. This work reveals a critical role for plant flotillins in symbiotic bacterial infection.

Abstract

The Sinorhizobium meliloti ExoS/ChvI two-component signaling pathway is required for the development of a nitrogen-fixing symbiosis between S. meliloti and its plant hosts. ExoS/ChvI also has important roles in regulating succinoglycan production, biofilm formation, motility, nutrient utilization, and the viability of free-living bacteria. Previous microarray experiments with an exoS96::Tn5 mutant indicated that ExoS/ChvI influences the expression of a few hundred genes, complicating the investigation of which downstream genes respond directly or indirectly to ExoS/ChvI regulation. To focus our study of ExoS/ChvI transcriptional target genes, we performed transcriptional profiling with chvI gain-of-function and reduced-function strains. The chvI gain-of-function strain that we used contains a dominant gain-of-function chvI allele in addition to wild-type chvI. We identified genes that, relative to their expression level in the wild type, are both upregulated in the chvI gain-of-function strain and downregulated in the reduced-function strain or vice versa. Guided by this focused set of genes, we performed gel mobility shift assays and demonstrated that ChvI directly binds the intergenic regions upstream of ropB1, SMb21440, and SMc01580. Furthermore, DNase I footprint analysis of the region upstream of SMc01580 identified a specific DNA sequence bound by ChvI and allowed the discovery of a possible motif for ChvI binding. Our results provide insight into the mechanism of how ExoS/ChvI regulates its downstream targets and lay a foundation for studying this conserved pathway with critical roles in free-living and symbiotic bacteria.

Abstract

Sinorhizobium meliloti is a symbiotic soil bacterium of the alphaproteobacterial subdivision. Like other rhizobia, S. meliloti induces nitrogen-fixing root nodules on leguminous plants. This is an ecologically and economically important interaction, because plants engaged in symbiosis with rhizobia can grow without exogenous nitrogen fertilizers. The S. meliloti-Medicago truncatula (barrel medic) association is an important symbiosis model. The S. meliloti genome was published in 2001, and the M. truncatula genome currently is being sequenced. Many new resources and data have been made available since the original S. meliloti genome annotation and an update was needed. In June 2008, we submitted our annotation update to the EMBL and NCBI databases. Here we describe this new annotation and a new web-based portal RhizoGATE. About 1000 annotation updates were made; these included assigning functions to 313 putative proteins, assigning EC numbers to 431 proteins, and identifying 86 new putative genes. RhizoGATE incorporates the new annotion with the S. meliloti GenDB project, a platform that allows annotation updates in real time. Locations of transposon insertions, plasmid integrations, and array probe sequences are available in the GenDB project. RhizoGATE employs the EMMA platform for management and analysis of transcriptome data and the IGetDB data warehouse to integrate a variety of heterogeneous external data sources.

Abstract

Sinorhizobium meliloti requires ExoS/ChvI two-component signalling to establish a nitrogen-fixing symbiosis with legume hosts. The importance of ExoS/ChvI signalling in microbe-host interactions is underscored by the requirement of ExoS/ChvI orthologues for virulence of the related alpha-proteobacteria Agrobacterium tumefaciens and Brucella abortus. In S. meliloti, ExoS/ChvI is a key regulator of gene expression for exopolysaccharide synthesis, biofilm formation, motility, nutrient utilization and free-living viability. Previously, we showed that the novel conserved regulator ExoR interacts genetically with both ExoS and ChvI, and localizes to the periplasm of S. meliloti. Here, we show that ExoR physically associates with ExoS and that this association is important for regulating ExoS/ChvI signalling. We have identified point mutations in the Sel1-like repeat region of ExoR that disrupt binding to ExoS and cause a dramatic increase in ExoS/ChvI-dependent gene expression. Furthermore, we have found that physical interaction with ExoS stabilizes the ExoR protein. Together, our results indicate that ExoR binds to ExoS in the periplasm of S. meliloti to inhibit ExoS/ChvI activity, and that ExoR represents a novel periplasmic inhibitor of two-component signalling.

Abstract

The plant hormone ethylene negatively regulates bacterial infection and nodule formation in legumes in response to symbiotic rhizobia, but the molecular mechanism(s) of ethylene action in symbiosis remain obscure. We have identified and characterized multiple mutant alleles of the MtSkl1 gene, which controls both ethylene sensitivity and nodule numbers. We show that this locus encodes the Medicago truncatula ortholog of the Arabidopsis ethylene signaling protein EIN2. In addition to the well-characterized role of MtSkl1 in rhizobial symbiosis, we show that MtSkl1 is involved in regulating early phases of the symbiotic interaction with mycorrhizal fungi, and in mediating root responses to cytokinin. MtSkl1 also functions in the defense against Rhizoctonia solani and Phytophthora medicaginis, with the latter interaction likely to involve positive feedback amplification of ethylene biosynthesis. Overexpression of the C-terminal domain of MtEIN2 is sufficient to block nodulation responses, consistent with previous reports in Arabidopsis on the activation of ethylene signaling. This same C-terminal region is uniquely conserved throughout the EIN2 homologs of angiosperms, which is consistent with its role as a higher plant-specific innovation essential to EIN2 function.

Abstract

screen for novel symbiotic mutants of the nitrogen-fixing legume symbiont Sinorhizobium meliloti uncovered a crucial role for the putative response regulator FeuP in the symbiotic infection process. Transcriptome analysis shows that FeuP controls the transcription of at least 16 genes, including ndvA, which encodes an ATP-dependent exporter of cyclic beta glucans. Loss of feuP function gives rise to traits associated with cyclic beta glucan biosynthetic defects, including poor growth and motility under hypoosmotic conditions, and the inability to invade plant tissue during the early stages of symbiotic infection. Analysis of cyclic glucans indicates that the feuP mutant is able to synthesize intracellular cyclic beta glucans, but is unable to export them. Cyclic beta glucan export can be restored to feuP mutant cells by constitutive expression of ndvA; likewise, the symbiotic phenotype of a feuP mutant is rescued by ectopic ndvA expression. We further show that the linked sensor kinase gene, feuQ, is also important for modulating ndvA transcription, and that signalling through the FeuP/FeuQ pathway is responsive to extracellular osmotic conditions, with low osmolarity stimulating ndvA expression.

Abstract

A large-scale screen for symbiotic mutants was carried out using the model root nodulating bacterium Sinorhizobium meliloti. Several mutations in the previously uncharacterized gene msbA2 were isolated. msbA2 encodes a member of the ATP-binding cassette exporter family. This protein family is known to export a wide variety of compounds from bacterial cells. S. meliloti MsbA2 is required for the invasion of nodule tissue, with msbA2 mutant cells stimulating nodule primordium morphogenesis, but failing to invade plant tissue beyond the epidermal cell layer. msbA2 mutants do not exhibit any of the free-living traits often found to correlate with symbiotic defects, suggesting that MsbA2 may take part in a specifically symbiotic function. In strains that overproduce the symbiotic signalling polysaccharide succinoglycan, loss of MsbA2 function is extremely deleterious. This synthetic lethal phenotype can be suppressed by disrupting the succinoglycan biosynthetic genes exoY or exoA. It can also be suppressed by disrupting putative glycosyltransferase-encoding genes found upstream of msbA2. Finally, the symbiotic phenotype of a msbA2 null mutant is suppressed by secondary mutations in these upstream transferase genes, indicating that the msbA2 mutant phenotype may be caused by an inhibitory accumulation of a novel polysaccharide that is synthesized from succinoglycan precursors.

Abstract

Sinorhizobium meliloti participates in a nitrogen-fixing symbiosis with legume plant host species of the genera Medicago, Melilotus, and Trigonella. We recently identified an S. meliloti two-component sensory histidine kinase, CbrA, which is absolutely required to establish a successful symbiosis with Medicago sativa (K. E. Gibson, G. R. Campbell, J. Lloret, and G. C. Walker, J. Bacteriol. 188:4508-4521, 2006). In addition to having a symbiotic defect, the cbrA::Tn5 mutant also has free-living phenotypes that suggest a cell envelope perturbation. Because the bases for these phenotypes are not well understood, we undertook an identification of CbrA-regulated genes. We performed a microarray analysis and compared the transcriptome of the cbrA::Tn5 mutant to that of the wild type. Our global analysis of gene expression identified 162 genes that are differentially expressed in the cbrA::Tn5 mutant, including those encoding proteins involved in motility and chemotaxis, metabolism, and cell envelope function. With regard to those genes with a known role in symbiosis, we observed increased expression of nine genes with overlapping functions in bacterial invasion of its host, which suggests that the mutant could be competent for invasion. Since these CbrA-repressed genes are vital to the invasion process, it appears that down-regulation of CbrA activity is important at this stage of nodule development. In contrast, our previous work showed that CbrA is required for bacteria to establish themselves within the host as nitrogen-fixing symbionts. Therefore, we propose a model in which CbrA functions as a developmental switch during symbiosis.

Abstract

The symbiotic association between legumes and nitrogen-fixing bacteria collectively known as rhizobia results in the formation of a unique plant root organ called the nodule. This process is initiated following the perception of rhizobial nodulation factors by the host plant. Nod factor (NF)-stimulated plant responses, including nodulation-specific gene expression, is mediated by the NF signaling pathway. Plant mutants in this pathway are unable to nodulate. We describe here the cloning and characterization of two mutant alleles of the Medicago truncatula ortholog of the Lotus japonicus and pea (Pisum sativum) NIN gene. The Mtnin mutants undergo excessive root hair curling but are impaired in infection and fail to form nodules following inoculation with Sinorhizobium meliloti. Our investigation of early NF-induced gene expression using the reporter fusion ENOD11::GUS in the Mtnin-1 mutant demonstrates that MtNIN is not essential for early NF signaling but may negatively regulate the spatial pattern of ENOD11 expression. It was recently shown that an autoactive form of a nodulation-specific calcium/calmodulin-dependent protein kinase is sufficient to induce nodule organogenesis in the absence of rhizobia. We show here that MtNIN is essential for autoactive calcium/calmodulin-dependent protein kinase-induced nodule organogenesis. The non-nodulating hcl mutant has a similar phenotype to Mtnin, but we demonstrate that HCL is not required in this process. Based on our data, we suggest that MtNIN functions downstream of the early NF signaling pathway to coordinate and regulate the correct temporal and spatial formation of root nodules.

Abstract

Sinorhizobium meliloti enters into a symbiotic relationship with legume host plants, providing fixed nitrogen in exchange for carbon and amino acids. In S. meliloti, exoR and the exoS-chvI two-component system regulate the biosynthesis of succinoglycan, an exopolysaccharide important for host invasion. It was previously reported that a loss-of-function mutation in exoR and a gain-of-function mutation in exoS cause overproduction of succinoglycan and loss of motility, indicating that ExoR negatively regulates and ExoS-ChvI positively regulates downstream genes. However, a relationship between exoR and exoS-chvI has never been clearly established. By identification and detailed characterization of suppressor strains, we provide genetic evidence that exoR and exoS-chvI control many similar phenotypes. These include succinoglycan production, symbiosis, motility, and previously uncharacterized prototrophy and biofilm formation, all of which are co-ordinately restored by suppressors. We further demonstrate that ExoR is located in the periplasm, suggesting that it functions to regulate downstream genes in a novel manner. In pathogenic bacteria closely related to S. meliloti, exoS-chvI homologues are required for virulence and the regulation of cell envelope composition. Our data suggest that periplasmically localized ExoR and ExoS-ChvI function together in a unique and critical regulatory system associated with both free-living and symbiotic states of S. meliloti.

Abstract

Rhizobial bacteria activate the formation of nodules on the appropriate host legume plant, and this requires the bacterial signaling molecule Nod factor. Perception of Nod factor in the plant leads to the activation of a number of rhizobial-induced genes. Putative transcriptional regulators in the GRAS family are known to function in Nod factor signaling, but these proteins have not been shown to be capable of direct DNA binding. Here, we identify an ERF transcription factor, ERF Required for Nodulation (ERN), which contains a highly conserved AP2 DNA binding domain, that is necessary for nodulation. Mutations in this gene block the initiation and development of rhizobial invasion structures, termed infection threads, and thus block nodule invasion by the bacteria. We show that ERN is necessary for Nod factor-induced gene expression and for spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase, DMI3, which is a component of the Nod factor signaling pathway. We propose that ERN is a component of the Nod factor signal transduction pathway and functions downstream of DMI3 to activate nodulation gene expression.

Abstract

NodD1 is a member of the NodD family of LysR-type transcriptional regulators that mediates the expression of nodulation (nod) genes in the soil bacterium Sinorhizobium meliloti. Each species of rhizobia establishes a symbiosis with a limited set of leguminous plants. This host specificity results in part from a NodD-dependent upregulation of nod genes in response to a cocktail of flavonoids in the host plant's root exudates. To demonstrate that NodD is a key determinant of host specificity, we expressed nodD genes from different species of rhizobia in a strain of S. meliloti lacking endogenous NodD activity. We observed that nod gene expression was initiated in response to distinct sets of flavonoid inducers depending on the source of NodD. To better understand the effects of flavonoids on NodD, we assayed the DNA binding activity of S. meliloti NodD1 treated with the flavonoid inducer luteolin. In the presence of luteolin, NodD1 exhibited increased binding to nod gene promoters compared to binding in the absence of luteolin. Surprisingly, although they do not stimulate nod gene expression in S. meliloti, the flavonoids naringenin, eriodictyol, and daidzein also stimulated an increase in the DNA binding affinity of NodD1 to nod gene promoters. In vivo competition assays demonstrate that noninducing flavonoids act as competitive inhibitors of luteolin, suggesting that both inducing and noninducing flavonoids are able to directly bind to NodD1 and mediate conformational changes at nod gene promoters but that only luteolin is capable of promoting the downstream changes necessary for nod gene induction.

Abstract

The Rhizobium-legume symbiosis culminates in the exchange of nutrients in the root nodule. Bacteria within the nodule reduce molecular nitrogen for plant use and plants provide bacteria with carbon-containing compounds. Following the initial signaling events that lead to plant infection, little is known about the plant requirements for establishment and maintenance of the symbiosis. We screened 44,000 M2 plants from fast neutron-irradiated Medicago truncatula seeds and isolated eight independent mutant lines that are defective in nitrogen fixation. The eight mutants are monogenic and represent seven complementation groups. To monitor bacterial status in mutant nodules, we assayed Sinorhizobium meliloti symbiosis gene promoters (nodF, exoY, bacA, and nifH) in the defective in nitrogen fixation mutants. Additionally, we used an Affymetrix oligonucleotide microarray to monitor gene expression changes in wild-type and three mutant plants during the nodulation process. These analyses suggest the mutants can be separated into three classes: one class that supports little to no nitrogen fixation and minimal bacterial expression of nifH; another class that supports no nitrogen fixation and minimal bacterial expression of nodF, bacA, and nifH; and a final class that supports low levels of both nitrogen fixation and bacterial nifH expression.

Abstract

Plant cell morphogenesis depends critically on two processes: the deposition of new wall material at the cell surface and the mechanical deformation of this material by the stresses resulting from the cell's turgor pressure. We developed a model of plant cell morphogenesis that is a first attempt at integrating these two processes. The model is based on the theories of thin shells and anisotropic viscoplasticity. It includes three sets of equations that give the connection between wall stresses, wall strains and cell geometry. We present an algorithm to solve these equations numerically. Application of this simulation approach to the morphogenesis of tip-growing cells illustrates how the viscoplastic properties of the cell wall affect the shape of the cell at steady state. The same simulation approach was also used to reproduce morphogenetic transients such as the initiation of tip growth and other non-steady changes in cell shape. Finally, we show that the mechanical anisotropy built into the model is required to account for observed patterns of wall expansion in plant cells.

Abstract

Rhizobial bacteria enter a symbiotic interaction with legumes, activating diverse responses in roots through the lipochito oligosaccharide signaling molecule Nod factor. Here, we show that NSP2 from Medicago truncatula encodes a GRAS protein essential for Nod-factor signaling. NSP2 functions downstream of Nod-factor-induced calcium spiking and a calcium/calmodulin-dependent protein kinase. We show that NSP2-GFP expressed from a constitutive promoter is localized to the endoplasmic reticulum/nuclear envelope and relocalizes to the nucleus after Nod-factor elicitation. This work provides evidence that a GRAS protein transduces calcium signals in plants and provides a possible regulator of Nod-factor-inducible gene expression.

A dual-genome Symbiosis Chip for coordinate study of signal exchange and development in a prokaryote-host interactionPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICABarnett, M. J., Tolman, C. J., Fisher, R. F., Long, S. R.2004; 101 (47): 16636-16641

Abstract

The soil-dwelling alpha-proteobacterium Sinorhizobium meliloti engages in a symbiosis with legumes: S. meliloti elicits the formation of plant root nodules where it converts dinitrogen to ammonia for use by the plant in exchange for plant photosynthate. To study the coordinate differentiation of S. meliloti and its legume partner during nodule development, we designed a custom Affymetrix GeneChip with the complete S. meliloti genome and approximately 10,000 probe sets for the plant host, Medicago truncatula. Expression profiling of free-living S. meliloti grown with the plant signal molecule luteolin in defined minimal and rich media or of strains altered in the expression of key regulatory proteins (NodD1, NodD3, and RpoN) confirms previous data and identifies previously undescribed regulatory targets. Analyses of root nodules show that this Symbiosis Chip allows the study of gene expression in both partners simultaneously. Our studies detail nearly 5,000 transcriptome changes in symbiosis and document complex transcriptional profiles of S. meliloti in different environments.

Abstract

Wall expansion in tip-growing cells shows variations according to position and direction. In Medicago truncatula root hairs, wall expansion exhibits a strong meridional gradient with a maximum near the pole of the cell. Root hair cells also show a striking expansion anisotropy, i.e. over most of the dome surface the rate of circumferential wall expansion exceeds the rate of meridional expansion. Concomitant measurements of expansion rates and wall stresses reveal that the extensibility of the cell wall must vary abruptly along the meridian of the cell to maintain the gradient of wall expansion. To determine the mechanical basis of expansion anisotropy, we compared measurements of wall expansion with expansion patterns predicted from wall structural models that were either fully isotropic, transversely isotropic, or fully anisotropic. Our results indicate that a model based on a transversely isotropic wall structure can provide a good fit of the data although a fully anisotropic model offers the best fit overall. We discuss how such mechanical properties could be controlled at the microstructural level.

Six nonnodulating plant mutants defective for Nod factor-induced transcriptional changes associated with the legume-rhizobia symbiosisPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMitra, R. M., Shaw, S. L., Long, S. R.2004; 101 (27): 10217-10222

Abstract

As the legume-rhizobia symbiosis is established, the plant recognizes bacterial-signaling molecules, Nod factors (NFs), and initiates transcriptional and developmental changes within the root to allow bacterial invasion and the construction of a novel organ, the nodule. Plant mutants defective in nodule initiation (Nod(-)) are thought to have defects in NF-signal transduction. However, it is unknown whether WT plants respond to NF-independent bacterial-derived signals or whether Nod(-) plant mutants show defects in global symbiosis-associated gene expression. To characterize plant gene expression in the establishment of the symbiosis, we used an Affymetrix oligonucleotide microarray representing 9,935 Medicago truncatula expressed sequences. We identified 46 sequences that are differentially expressed in plants exposed for 24 h to WT Sinorhizobium meliloti or to the invasion defective S. meliloti mutant, exoA. Eight of these genes encode nucleolar proteins, which are implicated in ribosome biogenesis. We also identified differentially expressed transcription factors, signaling components, defense response proteins, stress response proteins, and several previously uncharacterized genes. NF appears both necessary and sufficient to induce most changes. Six of seven Nod(-) M. truncatula mutants (nfp, dmi1, dmi2, dmi3, nsp1, and nsp2) showed no transcriptional response to S. meliloti, suggesting that the encoded proteins are required for initiating new transcription. The Nod(-) mutant hcl, however, exhibits a reduced transcriptional response to S. meliloti, indicating that the machinery responsible for initiating new transcription is at least partially functional in this mutant.

Abstract

In the establishment of the legume-rhizobial symbiosis, bacterial lipochitooligosaccharide signaling molecules termed Nod factors activate the formation of a novel root organ, the nodule. Nod factors elicit several responses in plant root hair cells, including oscillations in cytoplasmic calcium levels (termed calcium spiking) and alterations in root hair growth. A number of plant mutants with defects in the Nod factor signaling pathway have been identified. One such Medicago truncatula mutant, dmi3, exhibits calcium spiking and root hair swelling in response to Nod factor, but fails to initiate symbiotic gene expression or cell divisions for nodule formation. On the basis of these data, it is thought that the dmi3 mutant perceives Nod factor but fails to transduce the signal downstream of calcium spiking. Additionally, the dmi3 mutant is defective in the symbiosis with mycorrhizal fungi, indicating the importance of the encoded protein in multiple symbioses. We report the identification of the DMI3 gene, using a gene cloning method based on transcript abundance. We show that transcript-based cloning is a valid approach for cloning genes in barley, indicating the value of this technology in crop plants. DMI3 encodes a calcium/calmodulin-dependent protein kinase. Mutants in pea sym9 have phenotypes similar to dmi3 and have alterations in this gene. The DMI3 class of proteins is well conserved among plants that interact with mycorrhizal fungi, but it is less conserved in Arabidopsis thaliana, which does not participate in the mycorrhizal symbiosis.

Abstract

Legumes form symbiotic associations with both mycorrhizal fungi and nitrogen-fixing soil bacteria called rhizobia. Several of the plant genes required for transduction of rhizobial signals, the Nod factors, are also necessary for mycorrhizal symbiosis. Here, we describe the cloning and characterization of one such gene from the legume Medicago truncatula. The DMI1 (does not make infections) gene encodes a novel protein with low global similarity to a ligand-gated cation channel domain of archaea. The protein is highly conserved in angiosperms and ancestral to land plants. We suggest that DMI1 represents an ancient plant-specific innovation, potentially enabling mycorrhizal associations.

Abstract

In the Medicago truncatula/Sinorhizobium meliloti symbiosis, the plant undergoes a series of developmental changes simultaneously, creating a root nodule and allowing bacterial entry and differentiation. Our studies of plant genes reveal novel transcriptional regulation during the establishment of the symbiosis and identify molecular markers that distinguish classes of plant and bacterial symbiotic mutants. We have identified three symbiotically regulated plant genes encoding a beta,1-3 endoglucanase (MtBGLU1), a lectin (MtLEC4), and a cysteine-containing protein (MtN31). MtBGLU1 is down-regulated in the plant 24 h after exposure to the bacterial signal, Nod factor. The non-nodulating plant mutant dmi1 is defective in the ability to down-regulate MtBGLU1. MtLEC4 and MtN31 are induced 1 and 2 weeks after bacterial inoculation, respectively. We examined the regulation of these two genes and three previously identified genes (MtCAM1, ENOD2, and MtLB1) in plant symbiotic mutants and wild-type plants inoculated with bacterial symbiotic mutants. Plant (bit1, rit1, and Mtsym1) and bacterial (exoA and exoH) mutants with defects in the initial stages of invasion are unable to induce MtLEC4, MtN31, MtCAM1, ENOD2, and MtLB1. Bacterial mutants (fixJ and nifD) and a subset of plant mutants (dnf2, dnf3, dnf4, dnf6, and dnf7) defective for nitrogen fixation induce the above genes. The bacA bacterial mutant, which senesces upon deposition into plant cells, and two plant mutants with defects in nitrogen fixation (dnf1 and dnf5) induce MtLEC4 and ENOD2 but not MtN31, MtCAM1, or MtLB1. These data suggest the presence of at least three transcriptionally distinct developmental stages during invasion of M. truncatula by S. meliloti.

Abstract

The nitrogen-fixing symbiosis between Sinorhizobium meliloti and Medicago sativa requires complex physiological adaptation by both partners. One method by which bacteria coordinately control physiological adaptation is the stringent response, which is triggered by the presence of the nucleotide guanosine tetraphosphate (ppGpp). ppGpp, produced by the RelA enzyme, is thought to bind to and alter the ability of RNA polymerase (RNAP) to initiate and elongate transcription and affect the affinity of the core enzyme for various sigma factors. An S. meliloti relA mutant which cannot produce ppGpp was previously shown to be defective in the ability to form nodules. This mutant also overproduces a symbiotically necessary exopolysaccharide called succinoglycan. The work presented here encompasses the analysis of suppressor mutants, isolated from host plants, that suppress the symbiotic defects of the relA mutant. All suppressor mutations are extragenic and map to either rpoB or rpoC, which encode the beta and beta' subunits of RNAP. Phenotypic, structural, and gene expression analyses reveal that suppressor mutants can be divided into two classes; one is specific in its effect on stringent response-regulated genes and shares striking similarity with suppressor mutants of Escherichia coli strains that lack ppGpp, and another reduces transcription of all genes tested in comparison to that in the relA parent strain. Our findings indicate that the ability to successfully establish symbiosis is tightly coupled with the bacteria's ability to undergo global physiological adjustment via the stringent response.

Abstract

Establishment of the Rhizobium-legume symbiosis depends on a molecular dialogue, in which rhizobial nodulation (Nod) factors act as symbiotic signals, playing a key role in the control of specificity of infection and nodule formation. Using nodulation-defective (Nod-) mutants of Medicago truncatula to study the mechanisms controlling Nod factor perception and signalling, we have previously identified five genes that control components of a Nod factor-activated signal transduction pathway. Characterisation of a new M. truncatula Nod- mutant led to the identification of the Nod Factor Perception (NFP) locus. The nfp mutant has a novel phenotype among Nod- mutants of M. truncatula, as it does not respond to Nod factors by any of the responses tested. The nfp mutant thus shows no rapid calcium flux, the earliest detectable Nod factor response of wild-type plants, and no root hair deformation. The nfp mutant is also deficient in Nod factor-induced calcium spiking and early nodulin gene expression. While certain genes controlling Nod factor signal transduction also control the establishment of an arbuscular mycorrhizal symbiosis, the nfp mutant shows a wild-type mycorrhizal phenotype. These data indicate that the NFP locus controls an early step of Nod factor signal transduction, upstream of previously identified genes and specific to nodulation.

Abstract

Legumes and rhizobium bacteria form a symbiosis that results in the development of nitrogen-fixing nodules on the root of the host plant. The earliest plant developmental changes are triggered by bacterially produced nodulation (Nod) factors. Within minutes of exposure to Nod factors, sharp oscillations in cytoplasmic calcium levels (calcium spiking) occur in epidermal cells of several closely related legumes. We found that Lotus japonicus, a legume that follows an alternate developmental pathway, responds to both its bacterial partner and to the purified bacterial signal with calcium spiking. Thus, calcium spiking is not restricted to a particular pathway of nodule development and may be a general component of the response of host legumes to their bacterial partner. Using Nod factor-induced calcium spiking as a tool to identify mutants blocked early in the response to Nod factor, we show that the L. japonicus Ljsym22-1 mutant but not the Ljsym30 mutant fails to respond to Nod factor with calcium spiking.

Abstract

Bacterially derived Nod factor is critical in the establishment of the legume/rhizobia symbiosis. Understanding the mechanisms of Nod factor perception and signal transduction in the plant will greatly advance our understanding of this complex interaction. Here, we describe the identification of a new locus, nodulation-signaling pathway 2 (NSP2), of Medicago truncatula that is involved in Nod factor signaling. Mutants at this locus are blocked for Nod factor-induced gene expression and show a reduced root hair deformation response. nsp2 plants also show a complete absence of infection and cortical cell division following Sinorhizobium meliloti inoculation. Nod factor-induced calcium spiking, one of the earliest responses tested, is still functional in these mutant plants. We conclude that the gene NSP2 is a component of the Nod factor signal transduction pathway that lies downstream of the calcium-spiking response.

Abstract

Modulation of intracellular calcium levels plays a key role in the transduction of many biological signals. Here, we characterize early calcium responses of wild-type and mutant Medicago truncatula plants to nodulation factors produced by the bacterial symbiont Sinorhizobium meliloti using a dual-dye ratiometric imaging technique. When presented with 1 nM Nod factor, root hair cells exhibited only the previously described calcium spiking response initiating 10 min after application. Nod factor (10 nM) elicited an immediate increase in calcium levels that was temporally earlier and spatially distinct from calcium spikes occurring later in the same cell. Nod factor analogs that were structurally related, applied at 10 nM, failed to initiate this calcium flux response. Cells induced to spike with low Nod factor concentrations show a calcium flux response when Nod factor is raised from 1 to 10 nM. Plant mutants previously shown to be deficient for the calcium spiking response (dmi1 and dmi2) exhibited an immediate, truncated calcium flux with 10 nM Nod factor, demonstrating a competence to respond to Nod factor but an impaired ability to generate a full biphasic response. These results demonstrate that the legume root hair cell exhibits two independent calcium responses to Nod factor triggered at different agonist concentrations and suggests an early branch point in the Nod factor signal transduction pathway.

Abstract

We report the isolation and characterization of a new Medicago truncatula hyper-nodulation mutant, designated sunn (super numeric nodules). Similar to the previously described ethylene-insensitive mutant sickle, sunn exhibits a 10-fold increase in the number of nodules within the primary nodulation zone. Despite this general similarity, these two mutants are readily distinguished based on anatomical, genetic, physiological, and molecular criteria. In contrast to sickle, where insensitivity to ethylene is thought to be causal to the hyper-nodulation phenotype (R.V. Penmetsa, D.R. Cook [1997] Science 275: 527-530), nodulation in sunn is normally sensitive to ethylene. Nevertheless, sunn exhibits seedling root growth that is insensitive to ethylene, although other aspects of the ethylene triple response are normal; these observations suggest that hormonal responses might condition the sunn phenotype in a manner distinct from sickle. The two mutants also differ in the anatomy of the nodulation zone: Successful infection and nodule development in sunn occur predominantly opposite xylem poles, similar to wild type. In sickle, however, both infection and nodulation occur randomly throughout the circumference of the developing root. Genetic analysis indicates that sunn and sickle correspond to separate and unlinked loci, whereas the sunn/skl double mutant exhibits a novel and additive super-nodulation phenotype. Taken together, these results suggest a working hypothesis wherein sunn and sickle define distinct genetic pathways, with skl regulating the number and distribution of successful infection events, and sunn regulating nodule organogenesis.

Abstract

The Rhizobium-legume symbiosis involves the formation of a novel plant organ, the nodule, in which intracellular bacteria reduce molecular dinitrogen in exchange for plant photosynthates. Nodule development requires a bacterial signal referred to as Nod factor, which in Sinorhizobium meliloti is a beta-(1,4)-linked tetramer of N-acetylglucosamine containing N-acyl and O-acetyl modifications at the nonreducing end and a critical 6-O-sulfate at the reducing end. This sulfate modification requires the action of three gene products: nodH, which catalyzes the sulfonyl transfer, and nodPQ, which produce the activated form of sulfate, 3'-phosphoadenosine-5'-phosphosulfate. It was previously reported that S. meliloti cell surface polysaccharides are also covalently modified by sulfate in a reaction dependent on NodPQ. We have further characterized this unique form of bacterial carbohydrate modification. Our studies have determined that one of the nodPQ mutant strains used in the initial study of sulfation of cell surface harbored a second unlinked mutation. We cloned the gene affected by this mutation (referred to as lps-212) and found it to be an allele of lpsL, a gene previously predicted to encode a UDP-glucuronic acid epimerase. We demonstrated that lpsL encoded a UDP-glucuronic acid epimerase activity that was reduced in the lps-212 mutant. The lps-212 mutation resulted in an altered lipopolysaccharide structure that was reduced in sulfate modification in vitro and in vivo. Finally, we determined that the lps-212 mutation resulted in a reduced ability to elicit the formation of plant nodules and by altered infection thread structures that aborted prematurely.

Abstract

In the Rhizobium-legume symbiosis, compatible partners recognize each other through an exchange of signals. Plant inducers act together with bacterial transcriptional activators, the NodD proteins, to regulate the expression of bacterial biosynthetic nodulation (nod) genes. These genes direct the synthesis of a lipochito-oligosaccharide signal called Nod factor (NF). NFs elicit an early host response, root hair calcium spiking, that is initiated in root hair cells within 15 min of NF or live Rhizobium inoculation. We used calcium spiking as an assay to compare two closely related strains of Sinorhizobium meliloti, Rm1021 and Rm2011, derived from the same field isolate. We found that the two strains show a kinetic difference in the calcium spiking assay: Rm1021 elicits calcium spiking in host root hairs as rapidly as purified NF, whereas Rm2011 shows a significant delay. This difference can be overcome by raising expression levels of either the NodD transcriptional activators or GroEL, a molecular chaperone that affects expression of the biosynthetic nod genes. We further demonstrate that the delay in triggering calcium spiking exhibited by Rm2011 is correlated with a reduced amount of nod gene expression compared with Rm1021. Therefore, calcium spiking is a useful tool in detecting subtle differences in bacterial gene expression that affect the early stages of the Rhizobium-legume symbiosis.

Abstract

Sinorhizobium meliloti, a gram-negative soil bacterium, forms a nitrogen-fixing symbiotic relationship with members of the legume family. To facilitate our studies of transcription in S. meliloti, we cloned and characterized the gene for the alpha subunit of RNA polymerase (RNAP). S. meliloti rpoA encodes a 336-amino-acid, 37-kDa protein. Sequence analysis of the region surrounding rpoA identified six open reading frames that are found in the conserved gene order secY (SecY)-adk (Adk)-rpsM (S13)-rpsK (S11)-rpoA (alpha)-rplQ (L17) found in the alpha-proteobacteria. In vivo, S. meliloti rpoA expressed in Escherichia coli complemented a temperature sensitive mutation in E. coli rpoA, demonstrating that S. meliloti alpha supports RNAP assembly, sequence-specific DNA binding, and interaction with transcriptional activators in the context of E. coli. In vitro, we reconstituted RNAP holoenzyme from S. meliloti alpha and E. coli beta, beta', and sigma subunits. Similar to E. coli RNAP, the hybrid RNAP supported transcription from an E. coli core promoter and responded to both upstream (UP) element- and Fis-dependent transcription activation. We obtained similar results using purified RNAP from S. meliloti. Our results demonstrate that S. meliloti alpha functions are conserved in heterologous host E. coli even though the two alpha subunits are only 51% identical. The ability to utilize E. coli as a heterologous system in which to study the regulation of S. meliloti genes could provide an important tool for our understanding and manipulation of these processes.

Abstract

In the Rhizobium-legume symbiosis, compatible bacteria and host plants interact through an exchange of signals: Host compounds promote the expression of bacterial biosynthetic nod (nodulation) genes leading to the production of a lipochito-oligosaccharide signal, the Nod factor (NF). The particular array of nod genes carried by a given species of Rhizobium determines the NF structure synthesized and defines the range of legume hosts by which the bacterium is recognized. Purified NF can induce early host responses even in the absence of live Rhizobium One of the earliest known host responses to NF is an oscillatory behavior of cytoplasmic calcium, or calcium spiking, in root hair cells, initially observed in Medicago spp. and subsequently characterized in four other genera (D.W. Ehrhardt, R. Wais, S.R. Long [1996] Cell 85: 673-681; S.A. Walker, V. Viprey, J.A. Downie [2000] Proc Natl Acad Sci USA 97: 13413-13418; D.W. Ehrhardt, J.A. Downie, J. Harris, R.J. Wais, and S.R. Long, unpublished data). We sought to determine whether live Rhizobium trigger a rapid calcium spiking response and whether this response is NF dependent. We show that, in the Sinorhizobium meliloti-Medicago truncatula interaction, bacteria elicit a calcium spiking response that is indistinguishable from the response to purified NF. We determine that calcium spiking is a nod gene-dependent host response. Studies of calcium spiking in M. truncatula and alfalfa (Medicago sativa) also uncovered the possibility of differences in early NF signal transduction. We further demonstrate the sufficiency of the nod genes for inducing calcium spiking by using Escherichia coli BL21 (DE3) engineered to express 11 S. meliloti nod genes.

Abstract

Sinorhizobium meliloti and host legumes enter into a nitrogen-fixing, symbiotic relationship triggered by an exchange of signals between bacteria and plant. S. meliloti produces Nod factor, which elicits the formation of nodules on plant roots, and succinoglycan, an exopolysaccharide that allows for bacterial invasion and colonization of the host. The biosynthesis of these molecules is well defined, but the specific regulation of these compounds is not completely understood. Bacteria control complex regulatory networks by the production of ppGpp, the effector molecule of the stringent response, which induces physiological change in response to adverse growth conditions and can also control bacterial development and virulence. Through detailed analysis of an S. meliloti mutant incapable of producing ppGpp, we show that the stringent response is required for nodule formation and regulates the production of succinoglycan. Although it remains unknown whether these phenotypes are connected, we have isolated suppressor strains that restore both defects and potentially identify key downstream regulatory genes. These results indicate that the S. meliloti stringent response has roles in both succinoglycan production and nodule formation and, more importantly, that control of bacterial physiology in response to the plant and surrounding environment is critical to the establishment of a successful symbiosis.

Abstract

In the early stages of symbiosis between the soil bacterium Sinorhizobium meliloti and its leguminous host plant, alfalfa, bacterial nodulation (nod) genes are controlled by NodD1, NodD2, and NodD3, members of the LysR family of transcriptional regulators, in response to flavonoid and other inducers released by alfalfa. To gain an understanding of the biochemical aspects of this action, epitope-tagged recombinant NodD1 and NodD3 were overexpressed in Escherichia coli. The DNA binding properties of the purified recombinant NodD proteins were indistinguishable from those of NodD isolated from S. meliloti. In addition, the E. coli GroEL chaperonin copurified with the recombinant NodD proteins. In this study, we showed that NodD proteins are in vitro substrates of the GroESL chaperonin system and that their DNA binding activity is modulated by GroESL. This confirmed the earlier genetic implication that the GroESL chaperonin system is essential for the function of these regulators. Increased DNA binding activity by NodD1 in the presence of luteolin confirmed that NodD1 is involved in recognizing the plant signal during the early stages of symbiosis.

Abstract

Nod factor is a critical signalling molecule in the establishment of the legume/rhizobial symbiosis. The Nod factor of Sinorhizobium meliloti carries O-sulphate, O-acetate and C16:2 N-acyl attachments that define its activity and host specificity. Here we assess the relative importance of these modifications for the induction of calcium spiking in Medicago truncatula. We find that Nod factor structures lacking the O-sulphate, structures lacking the O-acetate and N-acyl groups, and structures lacking the O-acetate combined with a C18:1 N-acyl group all show calcium spiking when applied at high concentrations. These calcium responses are blocked in dmi1 and dmi2 mutants, suggesting that they function through the Nod factor signal transduction pathway. The dmi3 mutant, which is proposed to function in the Nod factor signal transduction pathway downstream of calcium spiking, shows increased sensitivity to Nod factor. This increased sensitivity is only active with wild-type Nod factor and was not present when the plants were treated with mutant Nod factor structures. We propose that the Nod factor signal transduction pathway is under negative feedback regulation that is activated at or downstream of DMI3 and requires structural components of the Nod factor molecule for activity.

Abstract

Hybridization to a PCR product derived from conserved sigma-factor sequences led to the identification of two Sinorhizobium meliloti DNA segments that display significant sequence similarity to the family of rpoH genes encoding the sigma(32) (RpoH) heat-shock transcription factors. The first gene, rpoH1, complements an Escherichia coli rpoH mutation. Cells containing an rpoH1 mutation are impaired in growth at 37 degrees C under free-living conditions and are defective in nitrogen fixation during symbiosis with alfalfa. A plasmid-borne rpoH1-gusA fusion increases in expression upon entry of the culture into the stationary phase of growth. The second gene, designated rpoH2, is 42% identical to the S. meliloti rpoH1 gene. Cells containing an rpoH2 mutation have no apparent phenotype under free-living conditions or during symbiosis with the host plant alfalfa. An rpoH2-gusA fusion increases in expression during the stationary phase of growth. The presence of two rpoH-like sequences in S. meliloti is reminiscent of the situation in Bradyrhizobium japonicum, which has three rpoH genes.

Abstract

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti contains three replicons: pSymA, pSymB, and the chromosome. We report here the complete 1,354,226-nt sequence of pSymA. In addition to a large fraction of the genes known to be specifically involved in symbiosis, pSymA contains genes likely to be involved in nitrogen and carbon metabolism, transport, stress, and resistance responses, and other functions that give S. meliloti an advantage in its specialized niche.

Abstract

Legumes form a mutualistic symbiosis with bacteria collectively referred to as rhizobia. The bacteria induce the formation of nodules on the roots of the appropriate host plant, and this process requires the bacterial signaling molecule Nod factor. Although the interaction is beneficial to the plant, the number of nodules is tightly regulated. The gaseous plant hormone ethylene has been shown to be involved in the regulation of nodule number. The mechanism of the ethylene inhibition on nodulation is unclear, and the position at which ethylene acts in this complex developmental process is unknown. Here, we used direct and indirect ethylene application and inhibition of ethylene biosynthesis, together with comparison of wild-type plants and an ethylene-insensitive supernodulating mutant, to assess the effect of ethylene at multiple stages of this interaction in the model legume Medicago truncatula. We show that ethylene inhibited all of the early plant responses tested, including the initiation of calcium spiking. This finding suggests that ethylene acts upstream or at the point of calcium spiking in the Nod factor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. Furthermore, ethylene appears to regulate the frequency of calcium spiking, suggesting that it can modulate both the degree and the nature of Nod factor pathway activation.

Abstract

The scarcity of usable nitrogen frequently limits plant growth. A tight metabolic association with rhizobial bacteria allows legumes to obtain nitrogen compounds by bacterial reduction of dinitrogen (N2) to ammonium (NH4+). We present here the annotated DNA sequence of the alpha-proteobacterium Sinorhizobium meliloti, the symbiont of alfalfa. The tripartite 6.7-megabase (Mb) genome comprises a 3.65-Mb chromosome, and 1.35-Mb pSymA and 1.68-Mb pSymB megaplasmids. Genome sequence analysis indicates that all three elements contribute, in varying degrees, to symbiosis and reveals how this genome may have emerged during evolution. The genome sequence will be useful in understanding the dynamics of interkingdom associations and of life in soil environments.

Abstract

During development of the symbiotic soil bacterium Sinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. Regulatory genes controlling the early stages of this process have not been identified. As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S. meliloti. We show that the S. meliloti CtrA belongs to the CtrA-like family of response regulators found in several alpha-proteobacteria. In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions. ctrA is also an essential gene in S. meliloti, and it is expressed similarly to the autoregulated C. crescentus ctrA in that both genes have complex promoter regions which bind phosphorylated CtrA.

Abstract

The symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules on the roots of the host plant. The early stages of nodule formation are induced by bacteria via lipochitooligosaccharide signals known as Nod factors (NFs). These NFs are structurally specific for bacterium-host pairs and are sufficient to cause a range of early responses involved in the host developmental program. Early events in the signal transduction of NFs are not well defined. We have previously reported that Medicago sativa root hairs exposed to NF display sharp oscillations of cytoplasmic calcium ion concentration (calcium spiking). To assess the possible role of calcium spiking in the nodulation response, we analyzed M. truncatula mutants in five complementation groups. Each of the plant mutants is completely Nod- and is blocked at early stages of the symbiosis. We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. Another mutant, altered in the DMI3 gene, has a similar mutant phenotype to dmi1 and dmi2 mutants but displays normal calcium spiking. The calcium behavior thus implies that the DMI3 gene acts either downstream of calcium spiking or downstream of a common branch point for the calcium response and the later nodulation responses. Two additional mutants, altered in the NSP and HCL genes, which show root hair branching in response to NF, are normal for calcium spiking. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.

Abstract

Fluorescent microspheres were used as material markers to investigate the relative rates of cell surface expansion at the growing tips of Medicago truncatula root hairs. From the analysis of tip shape and microsphere movements, we propose three characteristic zones of expansion in growing root hairs. The center of the apical dome is an area of 1- to 2- microm diameter with relatively constant curvature and high growth rate. Distal to the apex is a more rapidly expanding region 1 to 2 microm in width exhibiting constant surges of off-axis growth. This middle region forms an annulus of maximum growth rate and is visible as an area of accentuated curvature in the tip profile. The remainder of the apical dome is characterized by strong radial expansion anisotropy where the meridional rate of expansion falls below the radial expansion rate. Data also suggest possible meridional contraction at the juncture between the apical dome and the cell body. The cell cylinder distal to the tip expands slightly over time, but only around the circumference. These data for surface expansion in the legume root hair provide new insight into the mechanism of tip growth and the morphogenesis of the root hair.

Abstract

During the Rhizobium-legume symbiosis, bacteria enter the cells of host plants and differentiate into nitrogen-fixing bacteroids. Recent mutant screens and expression studies have revealed bacterial genes involved in the developmental pathway and demonstrate how the genetic requirements can vary from one host-microbe system to another. Whether bacteroids are terminally differentiated cells is an ongoing debate and new experimental systems are required to address this issue.

Abstract

Microtubules are thought to be major determinants of plant morphogenesis, through effects on planes of cell division and on directions of differential cell expansion. In differentiation and redifferentiation processes, tubulin expression may prove a useful early indicator of cell activity. We examined the expression and localization of the pea alpha-tubulin gene TubA1 in situ and in transgenic alfalfa (Medicago sativa) to explore its use as a probe for plant development, and as a test case for correct developmental expression between two legume species commonly compared for studies of symbiosis with Rhizobium. The TubA1 mRNA was more abundant in root tips and immature leaves than in other tissues of pea. The promoter of TubA1 was fused to beta-glucuronidase (GUS) to analyze alpha-tubulin expression in transgenic alfalfa. Transient assays indicated that the TubA1 gene is transcribed at moderate levels compared to the cauliflower mosaic virus (CaMV) 35S promoter. Histochemical staining for GUS activity confirmed a correlation between TubA1 expression and cell division in nodules, roots and leaves. TubA1 promoter activity was first detected in the inner cortex of the root between 18 h and 24 h after spot inoculation with Rhizobium meliloti. Expression of a c-myc epitope fused to the carboxy-terminus of TubA1 resulted in an incorporation into the microtubular cytoskeleton, demonstrating the effectiveness of at least one epitope tag in creating functional tubulin fusions.

Abstract

We have cloned and sequenced three genes from Rhizobium meliloti (Sinorhizobium meliloti) that are involved in sulfate activation for cysteine biosynthesis. Two of the genes display homology to the Escherichia coli cysDN genes, which code for an ATP sulfurylase (EC 2.7.7.4). The third gene has homology to the E. coli cysH gene, a 3'-phosphoadenosine-5'-phosphosulfate (PAPS) reductase (EC 1.8.99.4), but has greater homology to a set of genes found in Arabidopsis thaliana that encode an adenosine-5'-phosphosulfate (APS) reductase. In order to determine the specificity of the R. meliloti reductase, the R. meliloti cysH homolog was histidine tagged and purified, and its specificity was assayed in vitro. Like the A. thaliana reductases, the histidine-tagged R. meliloti cysH gene product appears to favor APS over PAPS as a substrate, with a Km for APS of 3 to 4 microM but a Km for PAPS of >100 microM. In order to determine whether this preference for APS is unique to R. meliloti among members of the family Rhizobiaceae or is more widespread, cell extracts from R. leguminosarum, Rhizobium sp. strain NGR234, Rhizobium fredii (Sinorhizobium fredii), and Agrobacterium tumefaciens were assayed for APS or PAPS reductase activity. Cell extracts from all four species also preferentially reduce APS over PAPS.

Abstract

During the symbiosis between the bacterium Rhizobium meliloti and plants such as alfalfa, the bacteria elicit the formation of nodules on the roots of host plants. The bacteria infect the nodule, enter the cytoplasm of plant cells and differentiate into a distinct cell type called a bacteroid, which is capable of fixing atmospheric nitrogen. To discover bacterial genes involved in the infection and differentiation stages of symbiosis, we obtained genes expressed at the appropriate time and place in the nodule by identifying promoters that are able to direct expression of the bacA gene, which is required for bacteroid differentiation. We identified 230 fusions that are expressed predominantly in the nodule. Analysis of 23 sequences indicated that only three encode proteins known to be involved in the Rhizobium-legume symbiosis, six encode proteins with homology to proteins not previously associated with symbiosis, and 14 have no significant similarity to proteins of known function. Disruption of a locus that encodes a protein with homology to a cell adhesion molecule led to a defect in the formation of nitrogen-fixing nodules, resulting in an increased number of nitrogen-starved plants. Our isolation of a large number of nodule-expressed genes will help to open the intermediate stages of nodulation to molecular analysis.

Abstract

Rhizobium meliloti can occupy at least two distinct ecological niches; it is found in the soil as a free-living saprophyte, and it also lives as a nitrogen-fixing intracellular symbiont in root nodules of alfalfa and related legumes. One approach to understanding how R. meliloti alters its physiology in order to become an integral part of a developing nodule is to identify and characterize genes that are differentially expressed by bacteria living inside nodules. We used a screen to identify genes under the control of the R. meliloti regulatory protein NodD3, SyrM, or SyrA. These regulatory proteins are expressed by bacteria growing inside the root nodule. One gene isolated in this screen was mapped to pSymB and displayed complex regulation. The gene was downregulated by the syrA gene product and also by glucose and succinate. This gene, referred to as agpA, encodes a periplasmic binding protein that is most similar to proteins from the periplasmic oligopeptide binding protein family. It is likely that AgpA binds alpha-galactosides, because alpha-galactosides induce the expression of agpA, and agpA mutants cannot utilize or transport these sugars. Activity of an agpA::TnphoA fusion was downregulated by SyrA. Because syrA is known to be expressed at high levels in intracellular symbiotic R. meliloti and at low levels in the free-living bacteria, we propose that AgpA may belong to the class of gene products whose expression decreases when R. meliloti becomes an intracellular symbiont.

Abstract

Exopolysaccharides (EPS) are produced by a wide assortment of bacteria including plant pathogens and rhizobial symbionts. Rhizobium meliloti mutants defective in EPS production fail to invade alfalfa nodules. Production of EPS in R. meliloti is likely controlled at several levels. We have characterized a new gene of this regulatory circuit. syrA was identified by its ability to confer mucoid colony morphology and by its ability to suppress the colonial phenotype of an exoD mutant. Here we show that syrA encodes a 9-kD hydrophobic protein that has sequence similarity to two other EPS regulatory proteins: ExoX of Rhizobium NGR234 and R. meliloti, and Psi of R. leguminosarum bv. phaseoli. The syrA transcription start site lies 522 nucleotides upstream of a non-canonical TTG start codon. The syrA promoter region is similar to the promoter region of the nodulation regulatory protein, nodD3. We found that in free-living bacteria, syrA expression is activated by the regulatory locus, syrM, but not by nodD3. In planta, syrM is not required for expression of syrA. Instead, expression of the nitrogen fixation (nifHDKE) genes upstream of syrA plays a role. Specific and distinct sets of genetic controls may operate at different times during nodule invasion.

Abstract

The Rhizobium meliloti SyrM protein activates transcription of nodD3 and syrA. Regulation of syrM is complex and may involve as yet undiscovered genes. Here we report the isolation of insertion mutants showing increased expression of a syrM-gusA gene fusion. Characterization of one mutant strain, designated SYR-B, revealed a mutation consisting of a transposon insertion linked to a large deletion. The corresponding wild-type DNA was cloned as a 5.3-kb BamHI fragment. Genetic and physical analysis of this DNA demonstrated that an open reading frame (ORF) near one end of the fragment, encoding the 16.5-kDa SyrB protein, is responsible for the repression of syrM activity. Results of complementation experiments with the 5.3-kb BamHI DNA led us to hypothesize that other genes within this DNA fragment interfere with the expression or activity of SyrB. Our analysis showed that the region upstream of syrB contains three ORFs. One ORF is similar to the Ros repressor of Agrobacterium tumefaciens and the MucR repressor of R. meliloti.

Abstract

A gene encoding a variant of green fluorescent protein (GFP) of Aequorea victoria was put under the control of a promoter which is constitutive in Rhizobium meliloti. The heterologous GFP gene was expressed at high levels during all stages of symbiosis, allowing R. meliloti cells to be visualized as they grew in the rhizosphere, on the root surface, and inside infection threads. In addition, nodules that were infected with bacteria which were synthesizing GFP fluoresced when illuminated with blue light. GFP-tagged bacteria could be seen inside infection threads, providing the opportunity to measure the growth rate and determine the patterns of growth of R. meliloti residing inside its host plant.

Abstract

Efficient establishment of the symbiosis between rhizobia and their host plants requires precise regulation of bacterial nod genes. The nod gene transcripts in Rhizobium meliloti have approximately 200 nucleotides of untranslated sequence 5' of the start codon (5' UTR). We measured the significance of this region by constructing fusions between deletion derivatives of nodF and the reporter beta-glucuronidase (GUS). Flavonoid-inducible expression of the fusions in R. meliloti was evident when extra copies of the positive transcriptional activators NodD1, NodD3, or SyrM were present. The fusions responded normally over a range of inducer concentrations in Rhizobium leguminosarum bv. trifolii. GUS assays in planta showed no significant difference between the deletion constructs and a wild-type fusion. We conclude that the 5' UTRs of the nod gene transcripts are unlikely to have a significant regulatory role.

Abstract

Rhizobium lipochitooligosaccharide signal molecules stimulate multiple responses in legume host plants, including changes in host gene expression, cell growth, and mitoses leading to root nodule development. The basis for signal transduction in the plant is not known. We examined cytoplasmic free calcium in host root hairs using calcium-sensitive reporter dyes. Image analysis of injected dyes revealed localized periodic spikes in cytoplasmic calcium levels that ensued after a characteristic lag following signal application. Structural features of the signal molecules required to cause nodulation responses in alfalfa are also essential for stimulating calcium spiking. A nonnodulating alfalfa mutant is defective in calcium spiking, consistent with the possibility that this mutant is blocked in an early stage of nodulation signal perception.

Abstract

In Rhizobium meliloti the syrM regulatory gene positively controls nod D3 and syrA, and nodD3 positively controls syrM and nod regulon genes such as nodABC, syrM and nodD3 are divergently transcribed and are separated by approximately 2.8 kb of DNA. The 885-bp SphI DNA fragment between syrM and nodD3 was subcloned and sequenced. Analysis of this intergenic region showed two open reading frames similar to those found in insertion elements of the IS3 family. We determined transcription initiation sites for both syrM and nodD3 using primer extension. The syrM transcription initiation site is 499 bp upstream of the syrM protein-coding region and downstream of a nod box which shows several differences from the R. meliloti nod box consensus sequence. We demonstrated binding of NodD3 to DNA containing the syrM nod box. The nodD3 start site maps 659 bp upstream of the nodD3 translation initiation site. A putative SyrM binding site was identified upstream of the nodD3 start site on the basis of sequence similarity to the upstream region of syrA, another locus regulated by SyrM.

CLONING AND CHARACTERIZATION OF THE SIGA GENE ENCODING THE MAJOR SIGMA-SUBUNIT OF RHIZOBIUM-MELILOTIJOURNAL OF BACTERIOLOGYRushing, B. G., Long, S. R.1995; 177 (23): 6952-6957

Abstract

Using PCR to create a probe based on conserved region 2 of sigma factors, we have cloned the sigA gene coding for the major sigma factor of Rhizobium meliloti. The 684-residue protein encoded by the sigA gene was expressed in vitro in coupled transcription-translation experiments with R. meliloti extracts and migrated aberrantly in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its deduced amino acid sequence is similar to that of RpoD of Escherichia coli and is nearly identical to that of SigA of the closely related bacterium Agrobacterium tumefaciens. Through Southern analysis, we located the gene on the R. meliloti main chromosome rather than on one of the megaplasmids. The sigA locus does not appear to be part of a macromolecular synthesis operon (MMS), as in many other bacterial species, but rather lies downstream of a partial open reading frame showing similarity to the threonine dehydrogenase gene (tdh) of E. coli.

Abstract

Early stages of nodulation involve the exchange of signals between the bacterium and the host plant. Bacterial nodulation (nod) genes are required for Rhizobium spp. to synthesize lipooligosaccharide morphogens, termed Nod factors. The common nod genes encode enzymes that synthesize the factor core structure, which is modified by host-specific gene products. Here we show direct in vitro evidence that Rhizobium meliloti NodH, a host-specific nodulation gene, catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to the terminal 6-O position of Nod factors, and we show substrate requirements for the reaction. Our results indicate that polymerization of the chitooligosaccharide backbone likely precedes sulfation and that sulfation is not absolutely dependent on the presence or the particular structure of the N-acyl modification. NodH sulfation provides a tool for the enzymatic in vitro synthesis of novel Nod factors, or putative Nod factors intermediates, with high specific activity.

Abstract

Rhizobium meliloti exists either as a free-living soil organism or as a differentiated endosymbiont bacteroid form within the nodules of its host plant, alfalfa (Medicago sativa), where it fixes atmospheric N2. Differentiation is accompanied by major changes in DNA replication and cell division. In addition, R. meliloti harbors three unique large circular chromosome-like elements whose replication coordination may be complex. As part of a study of DNA replication control in R. meliloti, we isolated a dnaA homolog. The deduced open reading frame predicts a protein of 57 kDa that is 36% identical to the DnaA protein of Escherichia coli, and the predicted protein was confirmed by immunoblot analysis. In a comparison with the other known DnaA proteins, this protein showed the highest similarity to that of Caulobacter crescentus and was divergent in some domains that are highly conserved in other unrelated species. The dnaA genes of a diverse group of bacteria are adjacent to a common set of genes. Surprisingly, analysis of the DNA sequence flanking dnaA revealed none of these genes, except for an rpsT homolog, also found upstream of dnaA in C. crescentus. Instead, upstream of rpsT lie homologs of fpg, encoding a DNA glycosylase, and fadB1, encoding an enoyl-coenzyme A hydratase with a strikingly high (53 to 55%) level of predicted amino acid identity to two mammalian mitochondrial homologs. Downstream of dnaA, there are two open reading frames that are probably expressed but are not highly similar to any genes in the databases. These results show that R. meliloti dnaA is located within a novel gene arrangement.

Abstract

The molecular chaperones related to GroEL (hsp60, cpn60) interact with partially folded proteins and appear to assist them to attain active and correctly folded conformation. They are required for cell viability but are probably more important for some processes than for others. Through a random genetic search to find loci that are required for expression of the Rhizobium meliloti nod (nodulation) genes, we isolated a mutant (B4) defective in luteolin-dependent activation of nod gene expression, and found it carries a Tn5 insertion within a chromosomal groEL gene (groELc) located just downstream of a groESc gene. The groELc mutation affected activity of three related LysR-type activator proteins NodD1, NodD3, and SyrM; on plants, the mutants formed nodules late, and the nodules were Fix-. Hybridization and protein expression analysis show that a similar groESL locus (groESLa) maps to the Rm1021 megaplasmid pSyma. Southern blot analysis revealed additional, but less closely related sequences hybridizing to groELc and groESc probes elsewhere in the R. meliloti genome. Clones of groESLc and groESLa can each restore robust phage lambda growth on an Escherichia coli groE mutant. Likewise each clone can complement all of the phenotypes observed for B4 mutants; thus, the two appear to be functionally equivalent if expression is controlled. We determined that groELc is required for normal DNA binding of the NodD target sequence in R. meliloti. GroEL coimmunopurifies with NodD1 from R. meliloti, which suggests a direct physical association between these proteins. GroEL is thus probably involved in the folding or assembly of transcriptionally active NodD.

THE PISUM-SATIVUM TUBA1 GENE, A MEMBER OF A SMALL FAMILY OF ALPHA-TUBULIN SEQUENCESPLANT MOLECULAR BIOLOGYBRIERLEY, H. L., Webster, P., Long, S. R.1995; 27 (4): 715-727

Abstract

alpha- and beta-tubulin proteins are subunits of microtubules, which as primary elements of the plant cytoskeleton play major roles in plant cell division and cell morphogenesis. Several higher-plant alpha- and beta-tubulin gene families have been reported to have at least six to nine members each. Using genomic Southern hybridizations and polymerase chain reaction (PCR) experiments, we have found that the Pisum sativum (garden pea) genome has only four copies of alpha-tubulin sequences and a similar number of beta-tubulin sequences. We have characterized the pea alpha-tubulin gene TubA1. Its nucleotide sequence predicts a 452 amino acid product which is 89-98% identical to those predicted for other plant alpha-tubulins. By S1 nuclease analysis we have located the transcript start site at 102 bases upstream of the ATG. We have also shown that the TubA1 gene is expressed by northern hybridization with a gene-specific probe.

Abstract

The nodulation genes nodP and nodQ are required for production of Rhizobium meliloti nodulation (Nod) factors. These sulfated oligosaccharides act as morphogenic signals to alfalfa, the symbiotic host of R. meliloti. In previous work, we have shown that nodP and nodQ encode ATP sulfurylase, which catalyzes the formation of APS (adenosine 5'-phosphosulfate) and PPi. In the subsequent metabolic reaction, APS is converted to PAPS (3'-phosphoadenosine 5'-phosphosulfate) by APS kinase. In Escherichia coli, cysD and cysN encode ATP sulfurylase; cysC encodes APS kinase. Here, we present genetic, enzymatic, and sequence similarity data demonstrating that nodP and nodQ encode both ATP sulfurylase and APS kinase activities and that these enzymes associate into a multifunctional protein complex which we designate the sulfate activation complex. We have previously described the presence of a putative GTP-binding site in the nodQ sequence. The present report also demonstrates that GTP enhances the rate of PAPS synthesis from ATP and sulfate (SO4(2-)) by NodP and NodQ expressed in E. coli. Thus, GTP is implicated as a metabolic requirement for synthesis of the R. meliloti Nod factors.

BIOSYNTHESIS OF RHIZOBIUM-MELILOTI LIPOOLIGOSACCHARIDE NOD FACTORS - NODA IS REQUIRED FOR AN N-ACYLTRANSFERASE ACTIVITYPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAAtkinson, E. M., Palcic, M. M., Hindsgaul, O., Long, S. R.1994; 91 (18): 8418-8422

Abstract

Rhizobium bacteria synthesize N-acylated beta-1,4-N-acetylglucosamine lipooligosaccharides, called Nod factors, which act as morphogenic signal molecules to legume roots during development of nitrogen-fixing nodules. The biosynthesis of Nod factors is genetically dependent upon the nodulation (nod) genes, including the common nod genes nodABC. We used the Rhizobium meliloti NodH sulfotransferase to prepare 35S-labeled oligosaccharides which served as metabolic tracers for Nod enzyme activities. This approach provides a general method for following chitooligosaccharide modifications. We found nodAB-dependent conversion of N-acetylchitotetraose (chitotetraose) monosulfate into hydrophobic compounds which by chromatographic and chemical tests were equivalent to acylated Nod factors. Sequential incubation of labeled intermediates with Escherichia coli containing either NodA or NodB showed that NodB was required before NodA during Nod factor biosynthesis. The acylation activity was sensitive to oligosaccharide chain length, with chitotetraose serving as a better substrate than chitobiose or chitotriose. We constructed a putative Nod factor intermediate, GlcN-beta 1,4-(GlcNAc)3, by enzymatic synthesis and labeled it by NodH-mediated sulfation to create a specific metabolic probe. Acylation of this oligosaccharide required only NodA. These results confirm previous reports that NodB is an N-deacetylase and suggest that NodA is an N-acyltransferase.

Abstract

We have identified a second homolog of the cell division gene, ftsZ, in the endosymbiont Rhizobium meliloti. The ftsZ2 gene was cloned by screening a genomic lambda library with a probe derived from PCR amplification of a highly conserved domain. It encodes a 36-kDa protein which shares a high level of sequence similarity with the FtsZ proteins of Escherichia coli and Bacillus subtilis and FtsZ1 (Z1) of R. meliloti but lacks the carboxy-terminal region conserved in other FtsZ proteins. The identity of the ftsZ2 gene product was confirmed both by in vitro transcription-translation in an R. meliloti S-30 extract and by overproduction in R. meliloti cells. As with Z1, the overproduction of FtsZ2 in E. coli inhibited cell division and induced filamentation, although to a lesser extent than with Z1. However, the expression of ftsZ2 in E. coli under certain conditions caused some cells to coil dramatically, a phenotype not observed during Z1 overproduction. Although several Tn3-GUS (glucuronidase) insertions in a plasmid-borne ftsZ2 gene failed to cross into the chromosome, one interruption in the chromosomal ftsZ2 gene was isolated, suggesting that ftsZ2 is nonessential for viability. The two ftsZ genes were genetically mapped to the R. meliloti main chromosome, approximately 100 kb apart.

Abstract

The development of nitrogen-fixing nodules is induced on the roots of legume host plants by Rhizobium bacteria. We employed a novel strategy to probe the underlying mechanism of nodule morphogenesis in alfalfa roots using pTZS, a broad host range plasmid carrying a constitutive trans-zeatin secretion (tzs) gene from Agrobacterium tumefaciens T37. This plasmid suppressed the Nod- phenotype of Rhizobium nodulation mutants such that mutants harboring pTZS stimulated the formation of nodulelike structures. Alfalfa roots formed more or fewer of these nodules according to both the nitrogen content of the environment and the position along the root at which the pTZS+ bacteria were applied, which parallels the physiological and developmental regulation of true Rhizobium nodule formation. This plasmid also conferred on Escherichia coli cells the ability to induce root cortical cell mitoses. Both the pattern of induced cell divisions and the spatially restricted expression of an alfalfa nodule-specific marker gene (MsENOD2) in pTZS-induced nodules support the conclusion that localized cytokinin production produces a phenocopy of nodule morphogenesis.

Abstract

We sequenced a small uncharacterized region in the Rhizobium meliloti nod gene cluster downstream of nodQ1. We found the beginning of a large open reading frame (260 amino acids) in this fragment. The sequence reported here has striking similarity to IS66 (Y. Machida, M. Sakurai, S. Kiyokawa, A. Ubasawa, and S. Yasushiro, 1984, Proc. Natl. Acad. Sci. USA 81:7495-7499), an insertion element found in an Agrobacterium tumefaciens mutant.

INTERACTIONS OF NODD AT THE NOD BOX - NODD BINDS TO 2 DISTINCT SITES ON THE SAME FACE OF THE HELIX AND INDUCES A BEND IN THE DNAJOURNAL OF MOLECULAR BIOLOGYFisher, R. F., Long, S. R.1993; 233 (3): 336-348

Abstract

The Rhizobium meliloti nodD gene products are positive transcriptional activators of genes required for early stages of nodule morphogenesis in the R. meliloti-alfalfa symbiosis (nod genes). The regulatory activity of NodD, a member of the LysR family of activator proteins, is mediated in part through its binding to conserved DNA sequences termed nod boxes which lie upstream of the inducible nod genes. Here we use interference footprinting to identify two NodD binding sites in the nodA, nodF and nodH nod boxes. These two binding sites are located on the same face of the DNA helix and can be separated by an additional 10 bp with retention of activity. By systematic alteration of the phasing of the two binding sites on the DNA helix, we showed that only constructs which contain both sites on the same side of the helix are recognized by NodD as determined by migration retardation assay and by in vivo activation of nod box-lacZ fusions. Moreover, NodD apparently induces a bend in the DNA upon binding at the nod box as shown by migration retardation behavior of circularly permuted nod box fragments.

Abstract

A 4-kb fragment active as an autonomously replicating sequence (ARS) from the Rhizobium meliloti symbiotic megaplasmid pSym-b was isolated by selecting for sequences that allowed a normally nonreplicative pBR322 derivative to replicate in R. meliloti. The resulting Escherichia coli-R. meliloti shuttle plasmid (mini-pSym-b) containing the ARS also replicated in the closely related Agrobacterium tumefaciens, but only in strains carrying pSym-b, suggesting that a megaplasmid-encoded trans-acting factor is required. The copy number of mini-pSym-b was approximately the same as that of the resident megaplasmid, and mini-pSym-b was unstable in the absence of antibiotic selection. An 0.8-kb DNA subfragment was sufficient for replication in both R. meliloti and A. tumefaciens. The minimal ARS exhibited several sequence motifs common to other replication origins, such as an AT-rich region, three potential DnA binding sites, a potential 13-mer sequence, and several groups of short direct repeats. Hybridization experiments indicated that there may be a related ARS on the other megaplasmid, pSym-a. The pSym-b ARS was mapped near exoA, within a region nonessential for pSym-b replication. These results suggest that the R. meliloti megaplasmids share conserved replication origins and that pSym-b contains multiple replication origins. Since the mini-pSym-b shuttle vector can coexist with IncP-1 broad-host-range plasmids, it is also now possible to use two compatible plasmids for cloning and genetic manipulation in R. meliloti.

Abstract

The early steps of symbiotic nodule formation by Rhizobium on plants require coordinate expression of several nod gene operons, which is accomplished by the activating protein NodD. Three different NodD proteins are encoded by Sym plasmid genes in Rhizobium meliloti, the alfalfa symbiont. NodD1 and NodD2 activate nod operons when Rhizobium is exposed to host plant inducers. The third, NodD3, is an inducer-independent activator of nod operons. We previously observed that nodD3 carried on a multicopy plasmid required another closely linked gene, syrM, for constitutive nod operon expression. Here, we show that syrM activates expression of the nodD3 gene, and that nodD3 activates expression of syrM. The two genes constitute a self-amplifying positive regulatory circuit in both cultured Rhizobium and cells within the symbiotic nodule. We find little effect of plant inducers on the circuit or on expression of nodD3 carried on pSyma. This regulatory circuit may be important for regulation of nod genes within the developing nodule.

Abstract

The nitrogen-fixing symbiont Rhizobium meliloti establishes nodules on leguminous host plants. Nodulation (nod) genes used for this process are located in a cluster on the pSym-a megaplasmid of R. meliloti. These genes include nodP and nodQ (here termed nodPQ), which encode ATP sulfurylase and APS kinase, enzymes that catalyze the conversion of ATP and SO(4)2- into the activated sulfate form 3'-phosphoadenosine 5'-phosphosulfate (PAPS), an intermediate in cysteine synthesis. In Rhizobium, PAPS is also a precursor for sulfated and N-acylated oligosaccharide Nod-factor signals that cause symbiotic responses on specific host plants such as alfalfa. We previously found a highly conserved second copy of nodPQ in R. meliloti. We report here the mapping and cloning of this second copy, and its location on the second megaplasmid, pSym-b. The function of nodP2Q2 is equivalent to that of nodP1Q1 in complementation tests of R. meliloti and Escherichia coli mutants in ATP sulfurylase and adenosine 5'-phosphosulfate (APS) kinase. Mutations in nodP2Q2 do not have as severe an effect on symbiosis or plant host range as do those in nodP1Q1, however, possibly reflecting differences in expression and/or channeling of metabolites to specific enzymes involved in sulfate transfer. Strains mutated or deleted for both copies of nodQ are severely defective in symbiotic phenotypes, but remain prototrophic. This suggests the existence in R. meliloti of a third locus for ATP sulfurylase and APS kinase activities. We have found a new locus saa (sulfur amino acid), which may also encode these activities.

Abstract

Rhizobium bacteria form nitrogen-fixing nodules on legume roots. As part of the nodulation process, they secrete Nod factors that are beta-1,4-linked oligomers of N-acetylglucosamine. These factors depend on nodulation (nod) genes, but most aspects of factor synthesis are not yet known. We show here that one gene, nodC, shows striking similarity to genes encoding proteins known to be involved in polysaccharide synthesis in yeast and bacteria, specifically chitin and cellulose synthases, as well as a protein with unknown function in Xenopus embryos, DG42. This similarity is consistent with a role for the NodC protein in the formation of the beta-1,4-linkage in Nod factors.

Abstract

Initial stages in the Rhizobium-legume symbiosis can be thought of as a reciprocal molecular conversation: transmission of a gene inducer from legume host to bacterium, with ensuing bacterial synthesis of a morphogen that is transmitted to the plant, switching the developmental fate of the legume root. These signal molecules have a key role in determining bacterium-host specificity and the purified Nod factor compounds provide useful new tools to probe plant cell function.

Abstract

Although much is known about the bacterial genetics of early nodulation, little is known about the plant cell response. Alfalfa root hair cells were impaled with intracellular microelectrodes to measure a membrane potential depolarizing activity in Rhizobium meliloti cell-free filtrates, a plant response dependent on the bacterial nodulation genes. The depolarization was desensitized by repeated exposure to factors and was not observed in a representative nonlegume. A purified extracellular Nod factor, NodRm-IV(S), caused membrane potential depolarization at nanomolar concentrations. This rapid single-cell assay provides a tool for dissecting the mechanisms of host cell response in early nodulation.

Abstract

The ftsZ gene is essential for initiation of cell division in Escherichia coli and Bacillus subtilis. To begin our studies of division arrest during differentiation of Rhizobium meliloti bacteroids, we isolated a R. meliloti ftsZ homolog, ftsZRm. Degenerate primers directed towards a conserved region of ftsZ were used to amplify a segment of R. meliloti DNA by polymerase chain reaction, and the product of this reaction was then used to isolate positive clones from a bacteriophage library. The DNA sequence of an open reading frame containing the region of homology indicated that the R. meliloti FtsZ protein (FtsZRm) is 50% homologous to the known E. coli and B. subtilis FtsZ proteins, but at 590 amino acids (63 kDa), it is predicted to be nearly 50% larger. Strong expression of an approximately 70-kDa labeled protein in a coupled in vitro transcription-translation system supports this prediction. The additional 200 amino acids appear to fall in a single internal domain highly enriched for proline and glutamine residues. When we regulated R. meliloti ftsZ (ftsZRm) expression on a high-copy-number plasmid in E. coli with Plac and laclq, cells were smaller than normal in the presence of low FtsZRm levels (with no isopropyl-beta-D-thiogalactopyranoside [IPTG]) and filamentous when FtsZRm was overproduced (with IPTG). These results suggest that low levels of FtsZRm stimulate E. coli cell division, while high levels may be inhibitory.

Abstract

Nodulation (nod) genes are required for invasion of legumes by Rhizobium bacteria. Mutant WL131 is a derivative of 102F51 that has a severe Nod- phenotype on alfalfa. Upon examination of the extended DNA region containing host-specific nodulation genes nodFEG and nodH, we found that the nodG gene of WL131 bears a novel insertion sequence, ISRm3. Complementation studies implied, however, that the phenotype on alfalfa correlated with the nodH locus. We found that nodH in WL131 encodes an altered gene product. Correlation of the WL131 defect with nodH was also supported by phenotypic behavior. Each mutation affected nodulation more severely on alfalfa (Medicago sativa) than on sweet clover (Melilotus albus). However, we found that the degree of requirement for nodH in nodulation varied with the conditions under which the plant was grown.

Abstract

The nodulation (nod) genes of the symbiont Rhizobium meliloti are transcriptionally controlled by protein activators in the nodD gene family. While NodD1 and NodD2 act in concert with small molecular weight inducers provided by the host legume plant, NodD3 is an inducer-independent activator of the nod promoters. We determined the sequence of the nodD3 gene, confirmed the expression of a 35 kDa protein in vitro, and determined the insertion points of five Tn5 insertions in the region of the nodD3 gene. We found the NodD3 amino acid sequence to be markedly diverged from the sequences of NodD1 and NodD2, which were more similar to the inducer-dependent NodD of another species, Rhizobium leguminosarum biovar viciae. The expression of nodD3 is not well understood, but involves at least SyrM, another positive activator related to the LysR-NodD family. One of the phenotypically mutant Tn5 insertions used in genetic studies of NodD3-dependent nod regulation lacks NodD3 protein as determined by Western blots, but another expresses about 50-60% of the wild type level. The location of these Tn5 insertions substantially upstream of the open reading frame for NodD3 suggests importance of relatively distant regulatory sequences for nodD3 expression. An insertion that did not cause a NodD3- phenotype is located in the extreme C-terminus of the protein coding region.

Abstract

The symbiotic bacterium Rhizobium meliloti stimulates alfalfa (Medicago sativa L.) roots to undergo morphogenesis and form nitrogen-fixing nodules. It has been proposed that the bacterial genes nodABC, common to all Rhizobium, are required for synthesis of an oligosaccharide factor, which is converted to a sulphated form (NodRm-1) by the products of the R. meliloti-specific genes nodH and nodQ1-5; NodRm-1 elicits host-specific plant responses. Previously we have shown that the nodP gene is homologous to a segment of the Escherichia coli genome; when we cloned this E. coli fragment we found that it mapped near 59 minutes, corresponding to the cysDNC locus. The genes cysD and cysN encode proteins that catalyse the synthesis of adenosine 5'-phosphosulphate, the first step in the activation of inorganic sulphate. Here we demonstrate that nodP and nodQ correspond to cysD and cysN, and that their proteins have ATP sulphurylase activity both in vivo and in vitro. We propose that nodP and nodQ synthesize an activated sulphate that is an intermediate in the formation of the alfalfa-specific sulphated nodRm-1 factor.

Abstract

Rhizobium meliloti nodulation (nod) genes are expressed when activated by trans-acting proteins in the NodD family. The nodD1 and nodD2 gene products activate nod promoters when cells are exposed to plant-synthesized signal molecules. Alternatively, the same nod promoters are activated by the nodD3 gene when nodD3 is carried in trans along with a closely linked global regulatory locus, syrM (symbiotic regulator) (J. T. Mulligan and S. R. Long, Genetics 122:7-18, 1989). In this article we report the nucleotide sequence of a 2.6-kilobase SphI fragment from R. meliloti SU47 containing syrM. Expression from this locus was confirmed by using in vitro transcription-translation assays. The open reading frame encoded a protein of either 33 or 36 kilodaltons whose sequence shows similarity to NodD regulatory proteins.

Abstract

The Rhizobium meliloti nodD1 and nodD3 gene products (NodD1 and NodD3) are members of the lysR-nodD gene regulator family. They are functionally distinct in that NodD1 transcriptionally activates other nod genes in the presence of a flavonoid inducer such as luteolin, while NodD3 is capable of activating nod gene expression at high levels in the absence of inducer. NodD1 and NodD3 are DNA-binding proteins which interact with DNA sequences situated upstream of the transcription initiation sites of at least three sets of inducible nod genes. We report the footprinting of NodD1- and NodD3-DNA complexes with both DNase I and the 1,10-phenanthroline-copper ion reagent. NodD1 and NodD3 both interacted with the nodABC, nodFE, and nodH promoters and protected from cleavage an extensive piece of DNA, including the nod box, from approximately -20 to -75 from the transcription start site for each of the three promoters. The constitutively activating protein NodD3 displayed an additional hypersensitive cleavage site in its footprint compared with NodD1.

Abstract

Previous studies had suggested the existence of nodulation (nod) genes downstream of nodG in Rhizobium meliloti strain 1021. We have established the DNA sequence and analyzed the translation products of the genes located in this position. Computer analysis of the DNA sequence revealed a number of overlapping putative open-reading frames (ORFs), so we constructed several clones that contained either full-length or truncated ORFs. The protein products of these clones were expressed in both R. meliloti and Escherichia coli in vitro transcription-translation systems. These assays unambiguously defined the expressed ORFs, which we named nodP and nodQ. In addition, we found homology to these genes, via Southern hybridizations, elsewhere in the genome of R. meliloti strain 1021, and in other species of Rhizobium. The nodP gene also displayed homology to E. coli. A computer search revealed significant homology between NodQ and the GDP binding domain of elongation factor Tu (EF-Tu).

Abstract

Nodulation (nod) gene expression in Rhizobium meliloti requires plant inducers and the activating protein product of the nodD gene. We have examined three genes in R. meliloti which have nodD activity and sequence homology. These three nodD genes are designated nodD1, nodD2 and nodD3, and have distinctive properties. The nodD1 gene product activates expression of the nodABC operon, as measured by a nodC-lacZ fusion or by transcript analysis, in the presence of crude seed or plant wash or the inducer, luteolin. The nodD3 gene product can cause a high basal (uninduced) level of nodC-lacZ expression and nodABC transcripts which is relatively unaffected by inducers. The effect of nodD3 is dependent on the presence of another gene, syrM (symbiotic regulator). By primer extension analysis we determined that the transcription start site is the same for nodD1 plus luteolin or nodD3-syrM mediated expression of nodA and nodH mRNAs. syrM also enhances the expression of another symbiotically important trait, production of extracellular polysaccharide. This regulatory effect of syrM requires locus syrA, which is linked to nodD3 and syrM. The syrM-syrA mediated increase in polysaccharide production requires at least some of the previously identified exo genes and may be a parallel regulatory event to the syrM-nodD3 control of nod promoters.

Abstract

The early events in the alfalfa-Rhizobium meliloti symbiosis include deformation of epidermal root hairs and the approximately concurrent stimulation of cell dedifferentiation and cell division in the root inner cortex. These early steps have been studied previously by analysis of R. meliloti mutants. Bacterial strains mutated in nodABC, for example, fail to stimulate either root hair curling or cell division events in the plant host, whereas exopolysaccharide (exo) mutants of R. meliloti stimulate host cell division but the resulting nodules are uninfected. As a further approach to understanding early symbiotic interactions, we have investigated the phenotype of a non-nodulating alfalfa mutant, MnNC-1008 (NN) (referred to as MN-1008). Nodulating and non-nodulating plants were inoculated with wild-type R. meliloti and scored for root hair curling and cell divisions. MN-1008 was found to be defective in both responses. Mutant plants inoculated with Exo- bacteria also showed no cell division response. Therefore, the genetic function mutated in MN-1008 is required for both root hair curling and cell division, as is true for the R. meliloti nodABC genes. These observations support the model that the distinct cellular processes of root hair curling and cell division are triggered by related mechanisms or components, or are causally linked.

Abstract

Using a plate induction assay, we demonstrate that alfalfa exudes inducer of Rhizobium meliloti nodulation genes. The inducer is exuded from the infectible zone of the root, accumulates to at least 1 micromolar, and is not affected by 10 millimolar nitrate. No zones of inhibition are observed. A nodulation minus mutant line of alfalfa, MN-1008, exudes normal levels of inducer. R. meliloti grown in rich medium requires ten-fold higher concentrations of luteolin to achieve half-maximal induction as compared to cells grown in a minimal medium. Flavonoids other than luteolin are found to have activity in R. meliloti nodulation gene induction assays. The compounds apigenin and eriodictyol have activities two-fifths and one-seventh that of luteolin, respectively. Several of the flavonoids tested (morin = naringenin > kaempferol = chrysin > quercetin = fisetin = hesperitin) demonstrate antagonistic activity toward induction by luteolin. The most effective antagonist is the coumarin, umbelliferone.

Abstract

Nodulation (nod) genes in Rhizobium meliloti are transcriptionally induced by flavonoid signal molecules, such as luteolin, produced by its symbiotic host plant, alfalfa. This induction depends on expression of nodD. Upstream of three inducible nod gene clusters, nodABC, nodFE, and nodH, is a highly conserved sequence referred to as a 'nod box.' The upstream sequences have no other obvious similarity. We have found that DNA fragments containing the regions upstream of all three inducible transcripts show altered electrophoretic mobility when treated with R. meliloti extracts. The ability of the extracts to interact specifically with these DNAs correlated with the genetic dosage of nodD1 or nodD3 and with the presence and concentration of the nodD1 or nodD3 protein (NodD1 or NodD3) in the extracts. Antiserum specific to NodD was used to construct an immunoaffinity column that permitted a substantial purification of NodD1; this preparation of NodD1 also displayed specific binding to restriction fragments containing DNA sequences found upstream of inducible nod genes. In addition, NodD-specific antiserum removed the specific DNA-binding activity from total Rhizobium cell extracts. The interaction of total extracts and of partially purified NodD protein with nod promoter sequences was competitive with an oligonucleotide representing the 3' 25-bp portion of the nod box. The interaction of R. meliloti extracts and NodD1 protein with nod gene upstream regions occurred independently of exposure of cells or extracts to flavone inducer.

Abstract

Rhizobium meliloti Nod(-) mutant WL131, a derivative of wild-type strain 102F51, was complemented by a clone bank of wild-type R. meliloti 1021 DNA, and clone pRmJT5 was recovered. Transfer of pRmJT5 conferred alfalfa nodulation on other Rhizobium species, indicating a role in host range determination for pRmJT5. Mutagenesis of pRmJT5 revealed several segments in which transposon insertion causes delay in nodulation, and/or marked reduction of the number of nodules formed on host alfalfa plants. The set of mutants indicated five regions in which nod genes are located; one mutant, nod-216, is located in a region not previously reported to encode a nodulation gene. Other mutant phenotypes correlated with the positions of open reading frames for nodH, nodF and nodE , and with a 2.2-kb EcoRI fragment. A mutant in nodG had no altered phenotype in this strain. One nodulation mutant was shown to be a large deletion of the common nod gene region. We present a discussion comparing the various studies made on this extended nod gene region.

Abstract

We have established the DNA sequence and analyzed the transcription and translation products of a series of putative nodulation (nod) genes in Rhizobium meliloti strain 1021. Four loci have been designated nodF, nodE, nodG and nodH. The correlation of transposon insertion positions with phenotypes and open reading frames was confirmed by sequencing the insertion junctions of the transposons. The protein products of these nod genes were visualized by in vitro expression of cloned DNA segments in a R. meliloti transcription-translation system. In addition, the sequence for nodG was substantiated by creating translational fusions in all three reading frames at several points in the sequence; the resulting fusions were expressed in vitro in both E. coli and R. meliloti transcription-translation systems. A DNA segment bearing several open reading frames downstream of nodG corresponds to the putative nod gene mutated in strain nod-216. The transcription start sites of nodF and nodH were mapped by primer extension of RNA from cells induced with the plant flavone, luteolin. Initiation of transcription occurs approximately 25 bp downstream from the conserved sequence designated the "nod box," suggesting that this conserved sequence acts as an upstream regulator of inducible nod gene expression. Its distance from the transcription start site is more suggestive of an activator binding site rather than an RNA polymerase binding site.

Abstract

Rhizobium meliloti nod genes are required for the infection of alfalfa. Induction of the nodC gene depends on a chemical signal from alfalfa and on nodD gene expression. By using a nodC-lacZ fusion, we have shown that the induction of the R. meliloti nodC gene and the expression of nodD occur at almost normal levels in other Rhizobium backgrounds and in Agrobacterium tumefaciens, but not in Escherichia coli. Xanthomonas campestris, or Pseudomonas savastanoi. Our results suggest that bacterial genes in addition to nodDABC are required for nod gene response to plant cells. We have found that inducing activity is present in other plant species besides alfalfa. Acetosyringone, the A. tumefaciens vir gene inducer, does not induce nodC.

Abstract

Nodulation genes in Rhizobium are required for invasion of the host plant. The nodABC operon is induced by plant activator molecules; this activation requires the gene product of the constitutively expressed nodD locus, which is transcribed divergently from nodABC. We are employing in vitro transcription to elucidate the molecular mechanism of nod gene activation. We used a micropurification technique to obtain RNA polymerase from Rhizobium meliloti, and here demonstrate that it initiated and terminated accurately at the Escherichia coli trp promoter-leader region. E. coli RNA polymerase, however, apparently fails to recognize R. meliloti promoters. We used the R. meliloti RNA polymerase in a minimal transcription system to attempt to localize the divergent start sites for nodD and nodABC. Transcript sizing and fingerprinting, together with synchronized single-round transcription experiments permit us to designate an in vitro transcription initiation site for nodD. Primer extension analysis of in vivo mRNA demonstrates that the initiation site which is utilized in vitro is the same site used in vivo. While nodABC is not transcribed in our minimal in vitro transcription system, this system should prove useful for the study of factors in induced cells which promote expression of this inducible promoter.

Abstract

The symbiotic interaction of Rhizobium meliloti and alfalfa results in the formation of nitrogen-fixing root nodules. Rhizobium meliloti nodABC genes are required for the early host responses of cortical cell divisions and root hair curling. The induction of nodABC expression by alfalfa exudates demonstrates host-symbiont signaling at an early stage in nodule development. The inducer molecule for nodABC expression was isolated from plant exudate by constructing a nodABC-lacZ fusion to monitor the inducing activity. From ultraviolet-visible absorption spectra, proton nuclear magnetic resonance, and mass spectrometry, the inducer was determined to be 3',4', 5,7-tetrahydroxyflavone (luteolin). Luteolin is a normal secondary plant metabolite found throughout the plant kingdom that may serve to control nodABC expression during nodule development. This regulatory role for a flavone contrasts with the function of some flavonoids as defense compounds.

INDUCTION OF RHOZOBIUM-MELILOTI NODC EXPRESSION BY PLANT EXUDATE REQUIRES NODDPROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICAMulligan, J. T., Long, S. R.1985; 82 (19): 6609-6613

Abstract

The soil bacterium Rhizobium meliloti invades and establishes a symbiosis with host plants such as alfalfa. Bacterial nodulation (nod) genes are required for this invasion, but their mechanism of action and the timing of their expression are not known. We have used translational lacZ fusions to monitor expression of nodD and nodC, which are located in the cluster of four nod genes on the R. meliloti megaplasmid (pSym). nodD is expressed at comparable levels by broth-grown bacterial cells and by cells exposed to exudates from aseptically grown plants. Activity of the nodC-lacZ protein fusion in broth-grown bacterial cells is very low. nodC-lacZ activity is increased approximately equal to 30-fold by plant exudate when nodD is expressed at a high level but not when nodD expression is low. Both fusions show differences in expression when borne on inc-P vectors as compared to when located on the pSym megaplasmid. nodD expression from vector-borne copies of the nod segment and response of nodC to plant exudate appear to require additional loci on the megaplasmid. Our results suggest that regulation of bacterial nod gene expression is an important control mechanism early in the symbiosis, and that the biochemical nature of some nod gene products may be cryptic except in cells grown in the presence of plant exudate.

Abstract

A set of conserved, or common, bacterial nodulation (nod) loci is required for host plant infection by Rhizobium meliloti and other Rhizobium species. Four such genes, nodDABC, have been indicated in R. meliloti 1021 by genetic analysis and DNA sequencing. An essential step toward understanding the function of these genes is to characterize their protein products. We used in vitro and maxicell Escherichia coli expression systems, together with gel electrophoresis and autoradiography, to detect proteins encoded by nodDABC. We facilitated expression of genes on these DNA fragments by inserting them downstream of the Salmonella typhimurium trp promoter, both in colE1 and incP plasmid-based vectors. Use of the incP trp promoter plasmid allowed overexpression of a nodABC gene fragment in R. meliloti. We found that nodA encodes a protein of 21 kilodaltons (kDa), and nodB encodes one of 28 kDa; the nodC product appears as two polypeptide bands at 44 and 45 kDa. Expression of the divergently read nodD yields a single polypeptide of 33 kDa. Whether these represent true Rhizobium gene products must be demonstrated by correlating these proteins with genetically defined Rhizobium loci. We purified the 21-kDa putative nodA protein product by gel electrophoresis, selective precipitation, and ion-exchange chromatography and generated antiserum to the purified gene product. This permitted the immunological demonstration that the 21-kDa protein is present in wild-type cells and in nodB- or nodC-defective strains, but is absent from nodA::Tn5 mutants, which confirms that the product expressed in E. coli is identical to that produced by R. meliloti nodA. Using antisera detection, we found that the level of nodA protein is increased by exposure of R. meliloti cells to plant exudate, indicating regulation of the bacterial nod genes by the plant host.

Abstract

Infection of alfalfa by the soil bacterium Rhizobium meliloti proceeds by deformation of root hairs and bacterial invasion of host tissue by way of an infection thread. We studied an 8.7-kilobase (kb) segment of the R. meliloti megaplasmid, which contains genes required for infection. Site-directed Tn5 mutagenesis was used to examine this fragment for nodulation genes. A total of 81 R. meliloti strains with mapped Tn5 insertions in the 8.7-kb fragment were evaluated for nodulation phenotype on alfalfa plants; 39 of the insertions defined a 3.5-kb segment containing nodulation functions. Of these 39 mutants, 37 were completely nodulation deficient (Nod-), and 2 at the extreme nif-distal end were leaky Nod-. Complementation analysis was performed by inoculating plants with strains carrying a genomic Tn5 at one location and a plasmid-borne Tn5 at another location in the 3.5-kb nodulation segment. Mutations near the right border of the fragment behaved as two distinct complementation groups. The segment in which these mutations are located was analyzed by DNA sequencing. Several open reading frames were found in this region, but the one most likely to function is 1,206 bases long, reading from left to right (nif distal to proximal) and spanning both mutation groups. The genetic behavior of this segment may be due either to the gene product having two functional domains or to a recombinational hot spot between the apparent complementation groups.

Abstract

Regions of the Rhizobium meliloti nodulation genes from the symbiotic plasmid were transferred to Agrobacterium tumefaciens and Rhizobium trifolii by conjugation. The A. tumefaciens and R. trifolii transconjugants were unable to elicit curling of alfalfa root hairs, but were able to induce nodule development at a low frequency. These were judged to be genuine nodules on the basis of cytological and developmental criteria. Like genuine alfalfa nodules, the nodules were initiated from divisions of the inner root cortical cells. They developed a distally positioned meristem and several peripheral vascular bundles. An endodermis separated the inner tissues of the nodule from the surrounding cortex. No infection threads were found to penetrate either root hairs or the nodule cells. Bacteria were found only in intercellular spaces. Thus, alfalfa nodules induced by A. tumefaciens and R. trifolii transconjugants carrying small nodulation clones of R. meliloti were completely devoid of intracellular bacteria. When these strains were inoculated onto white clover roots, small nodule-like protrusions developed that, when examined cytologically, were found to more closely resemble roots than nodules. Although the meristem was broadened and lacked a root cap, the protrusions had a central vascular bundle and other rootlike features. Our results suggest that morphogenesis of alfalfa root nodules can be uncoupled from infection thread formation. The genes encoded in the 8.7-kilobase nodulation fragment are sufficient in A. tumefaciens or R. trifolii backgrounds for nodule morphogenesis.

Abstract

Nodulation (nod) genes are required for Rhizobium meliloti to invade and stimulate nodule formation in its host, alfalfa. We have established the DNA sequence of nodD, nodA, and nodB, which are part of a gene cluster located 20 kb downstream of nifHDK on the R. meliloti pSym megaplasmid. The nodD open reading frame (308 amino acids) reads from proximal to nifHDK toward distal to nifHDK, divergently from nodA (196 aa) and nodB (217 aa). These two genes read from distal to nifHDK toward proximal, and are just upstream from the previously defined open reading frame for nodC. Fourteen Tn5 insertion sites have been sequenced in nodD, nodA, and nodB, revealing no major hotspots for insertion, but an overall preference for G/C bases at positions 1 and 9 of the 9-bp repeat.

Abstract

Generalized transduction of Rhizobium meliloti 1021 was carried out by bacteriophage N3. Genetic markers on the chromosome and the pSym megaplasmid were transduced, along with markers on several IncP plasmids. Cotransduction between transposon Tn5 insertions and integrated recombinant plasmid markers permitted correlation of cotransductional frequencies and known physical distances. Bacteriophage N3 was capable of infecting several commonly used strains of R. meliloti.

Abstract

A set of 19 symbiotic mutants of Rhizobium meliloti obtained by a Tn5 "suicide plasmid" mutagenesis procedure was characterized genetically and physically. As part of this characterization, we showed that R. meliloti strain 1021, like other R. meliloti strains, contains a very large indigenous plasmid (greater than 300 Md) that carries the structural genes for nitrogenase (nifHDK genes). Among the 19 symbiotic mutations studied, at least six were shown to reside on the megaplasmid. By a "walking procedure" we obtained from a cosmid clone bank a set of overlapping cosmids that contained megaplasmid sequences contiguous to nifHDK. A 90 kb region of contiguous DNA from these cosmids was used to probe the mutant strains for rearrangements within this region. The same six mutations that were located on the megaplasmid mapped within the 90 kb region examined, which included the structural genes for nitrogenase (nifHDK). A majority of the mutations characterized in this study could not be correlated with a bona fide Tn5 insertion into a symbiotic gene.

Abstract

We have physically and genetically characterized 20 symbiotic and 20 auxotrophic mutants of Rhizobium meliloti, the nitrogen-fixing symbiont of alfalfa (Medicago sativa), isolated by transposon Tn5 mutagenesis. A "suicide plasmid" mutagenesis procedure was used to generate TN-5-induced mutants, and both auxotrophic and symbiotic mutants were found at a frequency of 0.3% among strains containing random TN5 insertions. Two classes of symbiotic mutants were isolated: 4 of the 20 formed no nodules at all (Nod-), and 16 formed nodules which failed to fix nitrogen (Fix-). We used a combination of physical and genetic criteria to determine that in most cases the auxotrophic and symbiotic phenotypes could be correlated with the insertion of a single Tn5 elements. Once the Tn5 element was inserted into the R. meliloti genome, the frequency of its transposition to a new site was approximately 10-8 and the frequency of precise excision was less than 10-9. In approximately 25% of the mutant strains, phage Mu DNA sequences, which originated from the suicide plasmid used to generate the Tn5 transpositions, were also found in the R. meliloti genome contiguous with Tn5. These later strains exhibited anomalous conjugation properties, and therefore we could not correlate the symbiotic phenotype with a Tn5 insertion. In general, we found that both physical and genetic tests were required to fully characterize transposon-induced mutations.

Abstract

Alfalfa roots infected with four nodulation defective (Nod-) mutants of Rhizobium meliloti which were generated by transposon Tn5 mutagenesis were examined by light and electron microscopy. In one class of Nod- mutants, which we can nonreactive, the bacteria did not induce root hair curling or penetrate host cells. In a second class of Nod- mutants, which we call reactive, the bacteria induced some root hair curling and entered root epidermal cells, although no infection threads were formed. In addition, reactive Nod- mutants induced extensive root hair proliferation and hypertrophied roots. This study presents the details of the phenotype of the association between each mutant strain and alfalfa roots.

Abstract

We have constructed a cosmid derivative of the low copy-number broad host-range cloning vector pRK290 (Ditta et al., 1980) by inserting a 1.6-kb Bg/II fragment containing lambda cos into the unique Bg/II site in pRK290. The new vector, pLAFR1, is 21.6 kb long, confers tetracycline resistance, contains a unique EcoRI site, and can be mobilized into and stably replicates within many Gram-negative hosts. We constructed a clone bank of Rhizobium meliloti DNA in pLAFR1 using a partial EcoRI digest. The mean insert size was 23.1 kb. When the clone bank was mated (en masse) from Escherichia coli to various R. meliloti auxotrophic mutants, tetracycline-resistant (Tcr) transconjugants were obtained at frequencies ranging from 0.1 to 0.8, and among these, prototrophic colonies were obtained at frequencies ranging from 0.001 to 0.007. pLAFR1 cosmids were mobilized from R. meliloti prototrophic colonies into E. coli and then reintroduced into R. meliloti auxotrophs. In most cases, 100% of these latter Tcr transconjugants were prototrophic.

Abstract

After transposon Tn5 mutagenesis, a high proportion of Rhizobium meliloti symbiotic mutants do not contain Tn5 insertions in symbiotic genes. Instead, the mutations in these strains are correlated with the presence of an endogenous insertion sequence (ISRm1) in nitrogen fixation (nif) or symbiotic genes which are adjacent to the nif genes. ISRm1 is 1.4 kb and transposes to at least three restriction fragments in the nif region at a frequency between 10(-2) and 10(-3). A nif region restriction fragment containing ISRm1 was cloned from one of the mutant strains unable to fix nitrogen symbiotically (Fix-) and the resulting plasmid was used as a hybridization probe. ISRm1 is present at least ten times in the R. meliloti genome but is not present in any other R. meliloti strains, E. coli strains, or Rhizobium species tested. We demonstrated that the Fix- phenotype correlated with ISRm1 transposition is indeed caused by ISRm1 insertion by conjugating a cloned fragment containing ISRm1 into a wild type Fix+ R. meliloti host and replacing the normal genomic nif fragment with the nif::ISRm1 fragment. The resulting strain was Fix-.